Lysophosphatidylcholine compositions

ABSTRACT

The present invention provides marine lysophosphatidylcholine compositions for use in pharmaceuticals, nutraceuticals and functional foods, as well as methods for making marine lysophosphatidylcholine compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/229,031 filed Dec. 21, 2018, now allowed as U.S. Pat. No. 11,065,267,which claims the priority benefit of U.S. Provisional Application No.62/725,683 filed Aug. 31, 2018, and U.S. Provisional Application No.62/608,891, filed Dec. 21, 2017, which are incorporated herein byreference in their entireties.

FIELD OF THE INVENTION

The present invention provides marine lysophosphatidylcholinecompositions for use in pharmaceuticals, nutraceuticals and functionalfoods, as well as methods for making marine lysophosphatidylcholinecompositions.

BACKGROUND OF THE INVENTION

Lysophosphatidylcholine (LPC) is a compound resulting from partialhydrolysis of a phosphatidylcholine (PC) molecule so that one of thefatty acid groups attached to the PC molecule is removed.

Lysophosphatidylcholine, with one mole of fatty acid per mole of lipidin position sn-1, is found in trace amounts in most animal tissues (atgreater concentrations, it disrupts membranes). It is produced byhydrolysis of dietary and biliary phosphatidylcholine and is absorbed assuch in the intestines, but it is re-esterified before being exported inthe lymph. In addition, it is formed in most tissues by hydrolysis ofphosphatidylcholine by means of the superfamily of phospholipase A2enzymes as part of the de-acylation/re-acylation cycle that controls theoverall molecular species composition of the latter, as discussed above.In plasma of animal species, an appreciable amount oflysophosphatidylcholine is formed by the action of the enzymelecithin:cholesterol acyltransferase (LCAT), which is secreted from theliver. This catalyses the transfer of fatty acids from position sn-2 ofphosphatidylcholine to free cholesterol in plasma, with formation ofcholesterol esters and of course of lysophosphatidylcholine, whichconsists of a mixture of molecular species with predominately saturatedand mono- and dienoic fatty acid constituents. In plasma, it is bound toalbumin and lipoproteins so that its effective concentration is reducedto a safe level. Identification of a highly specific phospholipase A2γin peroxisomes that is unique in generating sn-2-arachidonoyllysophosphatidylcholine suggests that this may be of relevance toeicosanoid generation and signalling. Elevated levels of26:0-lysophosphatidylcholine in blood are reported to be characteristicof Zellweger spectrum disorders (the result of a defect in peroxisomebiogenesis).

Lysophosphatidylcholine has pro-inflammatory properties and it is knownto be a pathological component of oxidized lipoproteins (LDL) in plasmaand of atherosclerotic lesions; for example, there is reportedly aspecific enrichment of 2-arachidonoyl-lysophosphatidylcholine in carotidatheroma plaque from type 2 diabetic patients. Recently, it has beenfound to have some functions in cell signalling, and specific receptors(coupled to G proteins) have been identified. It activates the specificphospholipase C that releases diacylglycerols and inositol triphosphatewith resultant increases in intracellular Ca2+ and activation of proteinkinase C. It also activates the mitogen-activated protein kinase incertain cell types, and it promotes demyelination in the nervous system.In vascular endothelial cells, it induces the important pro-inflammatorymediator cyclooxygenase-2 (COX-2), a key enzyme in prostaglandinsynthesis. Elevated levels of lysophosphatidylcholine have beenidentified in cervical cancer and may be diagnostic for the disease.Some biological effects of lysophosphatidylcholine may be simply due toits ability to diffuse readily into membranes, altering their curvatureand indirectly affecting the properties of membrane proteins.

WO2015048554 discloses methods for identifying compounds that modulatetransport via the Mfsd2a protein and other potential uses of LPCcompositions.

SUMMARY OF THE INVENTION

The present invention provides marine lysophosphatidylcholinecompositions for use in pharmaceuticals, nutraceuticals and functionalfoods, as well as methods for making marine lysophosphatidylcholinecompositions.

Accordingly, in some preferred embodiments, the present inventionprovides marine lysophosphatidylcholine (LPC) compositions orconcentrates characterized in comprising from about 10% to about 100%LPC w/w of the composition and an omega-3 fatty acid content of from 5%to 50% w/w of the composition and optionally having one or more thefollowing characteristics or properties: a) a 2-LPC:1-LPC ratio of from1:8 to 18:1; b) a phosphatidylcholine (PC) content of less than 10% w/wof the composition; c) a phosphatidylethanolamine (PE) content of lessthan 1.2% w/w of the composition; d) a neutral lipid content of from 5%to 65% w/w of the composition; e) a 2-LPC ether content of less than1.0% w/w; and f) a ratio of EPA:DHA of from 1:1 to 3:1 or a ratio ofDHA:EPA of from 1:1 to 5:1.

In some preferred embodiments, the composition comprises from 60% to100% LPC w/w of the composition. In some preferred embodiments, thecomposition comprises from 70% to 90% LPC w/w of the composition. Insome preferred embodiments, the composition comprises from 20% to 50%LPC w/w of the composition. In some preferred embodiments, thecomposition comprises from 20% to 30% LPC w/w of the composition. Insome preferred embodiments, the composition comprises from 10% to 20%LPC w/w of the composition.

In some preferred embodiments, the composition has an omega-3 fatty acidcontent of from 30% to 50% w/w of the composition. In some preferredembodiments, the composition has an omega-3 fatty acid content of from35% to 45% w/w of the composition. In some preferred embodiments, thecomposition has an omega-3 fatty acid content of from 5% to 20% w/w ofthe composition.

In some preferred embodiments, the compositions have property (a). Insome preferred embodiments, the compositions have property (b). In somepreferred embodiments, the compositions have property (c). In somepreferred embodiments, the compositions have property (d). In somepreferred embodiments, the compositions have property (e). In somepreferred embodiments, the compositions have property (f). In somepreferred embodiments, the compositions have two or more of properties(a), (b) and (c). In some preferred embodiments, the compositions havetwo or more of properties (a), (b), (c), (d), (e) and (f). In somepreferred embodiments, the compositions have three or more of properties(a), (b), (c), (d), (e) and (f). In some preferred embodiments, thecompositions have four or more of properties (a), (b), (c), (d), (e) and(f). In some preferred embodiments, the compositions have five or moreof properties (a), (b), (c), (d), (e) and (f). In some preferredembodiments, the compositions have properties (a), (b), (c), (d), (e)and (f).

In some preferred embodiments, the compositions are selected from thegroup consisting of a krill LPC composition, a herring LPC composition,a herring roe LPC composition, an algal LPC composition, and a CalanusLPC composition.

In some preferred embodiments, the present invention provides apharmaceutical or nutraceutical composition comprising a composition asdescribed above and a physiologically acceptable carrier. In somepreferred embodiments, the physiologically acceptable carrier is a lipidcarrier.

In some preferred embodiments, the present invention provides an oraldelivery vehicle containing the marine LPC composition, pharmaceuticalcomposition or nutraceutical composition as described above.

In some preferred embodiments, the present invention provides a lipidcomposition comprising a first lipid fraction and second lipid fraction,wherein the first lipid fraction is a marine LPC composition asdescribed above and the second lipid fraction is obtained from adifferent source than the first lipid fraction and/or contains less than20% LPC. In some preferred embodiments, the second lipid fraction isselected from the group consisting of a triglyceride fraction, adiglyceride fraction, a fatty acid ethyl ester fraction, a free fattyacid fraction and combinations thereof. In some preferred embodiments,the second lipid fraction is a marine lipid fraction comprising EPAand/or DHA. In some preferred embodiments, the present inventionprovides a pharmaceutical or nutraceutical composition comprising thelipid composition as just described and a physiologically acceptablecarrier. In some preferred embodiments, the present invention providesan oral delivery vehicle containing the lipid composition as justdescribed.

In some preferred embodiments, the present invention provides methodsfor making a lysophosphatidylcholine (LPC) composition with a highcontent of EPA and DHA from a marine raw material containingphospholipids comprising treating the marine raw material with aphospholipase that is not native to the marine raw material to provide aphospholipase treated raw material and fractionating the phospholipasetreated raw material to provide a lipid composition having a higherlysophosphatidylcholine content than the starting raw material. In somepreferred embodiments, the raw material is selected from the groupconsisting of a krill lipid preparation, a herring lipid preparation, aherring roe lipid preparation, an algal lipid preparation, and a Calanuslipid preparation. In some preferred embodiments, the krill lipidpreparation is a Euphausia superba lipid preparation.

In some preferred embodiments, the raw material is contacted with aphospholipase in a solvent. In some preferred embodiments, the solventis a mixture of water and an alcohol. In some preferred embodiments, thealcohol is ethanol. In some preferred embodiments, the raw material iscontacted with a phospholipase in a mixture of about 85% water and 15%ethanol.

In some preferred embodiments, the enzyme is a phospholipase A1 (PLA1).In some preferred embodiments, the enzyme is a phospholipase A1 (PLA1),wherein the enzyme concentration is in the range of 0.1-20 vol/wt %,preferably 0.1-15 vol/wt %, more preferably 0.1-10 vol/wt %, furtherpreferably 0.1-5 vol/wt %, most preferably 0.1-3 vol/wt %. In somepreferred embodiments, the enzyme is a phospholipase A1 (PLA1), whereinthe method is carried out at a pH of 3-12, preferably 4-10, morepreferably 4-9, most preferably 5-9. In some preferred embodiments, theenzyme is a phospholipase A1 (PLA1), wherein the method is carried outbetween 4-95° C., preferably 4-85° C., more preferably 10-80° C.,further preferably 15-70° C., even more preferably 15-65° C., mostpreferably 15-60° C.

In some preferred embodiments, the raw material has a content of EPA andDHA at the range of; EPA: 1-70 wt % and DHA: 1-70 wt %, more preferablyEPA: 5-70 wt % and DHA: 5-70 wt %, most preferably EPA: 10-60 wt % andDHA: 10-60 wt %. In some preferred embodiments, the LPC composition hasa content of EPA and DHA in the range of; EPA: 1-100 wt % and/or DHA:1-100 wt %, more preferably EPA: 5-100 wt % and/or DHA: 5-100 wt %, mostpreferably EPA: 10-90 wt % and/or DHA: 10-90 wt %.

In some preferred embodiments, the LPC composition has an LPC content inthe ranges of 10-100 wt %, preferably 20-100 wt %, more preferably30-100 wt %, further preferably 40-100 wt %, most preferably 50-100 wt%. In some preferred embodiments, the LPC composition obtained by theprocess is characterized in comprising from about 20% to about 95% LPCw/w of the composition and an omega-3 fatty acid content of from 5% to50% w/w of the composition and optionally having one or more thefollowing characteristics or properties: a) a 2-LPC:1-LPC ratio of from1:8 to 18:1; b) a phosphatidylcholine (PC) content of less than 10% w/wof the composition; c) a phosphatidylethanolamine (PE) content of lessthan 1.2% w/w of the composition; d) a neutral lipid content of from 5%to 65% w/w of the composition; e) a 2-LPC ether content of less than1.0% w/w; and f) a ratio of EPA:DHA of from 1:1 to 3:1 or a DHA:EPAratio of 1:1 to 5:1.

In some preferred embodiments, the methods further comprise formulatingthe LPC composition for human consumption.

In some preferred embodiments, the present invention provides an LPC orlipid composition as described above, or made by a method as describedabove, for use to supplement the diet of a human subject, preferably ofless than 10 years of age, more preferably less than 1 year of age, evenmore preferably less than 1 month of age, and most preferably a newborn.

In some preferred embodiments, the present invention provides marinelysophosphatidylcholine (LPC) compositions or concentrates characterizedin comprising from about 60% to about 100% LPC w/w of the compositionand an omega-3 fatty acid content of from 30% to 50% w/w of thecomposition and optionally having one or more the followingcharacteristics or properties: a) a 2-LPC:1-LPC ratio of from 1:8 to18:1 or more preferably from 1:1 to 10:1; b) a phosphatidylcholine (PC)content of less than 0.5% to 5% w/w of the composition; c) aphosphatidylethanolamine (PE) content of less than 0.5% w/w of thecomposition; d) a neutral lipid content of from less than 5% to 40% w/wof the composition; and e) a ratio 2-LPC:2-LPC ether of from 2.5:1 to4:1.

In some preferred embodiments, the present invention provides marinelysophosphatidylcholine (LPC) compositions or concentrates characterizedin comprising from about 10% to about 20% LPC w/w of the composition andan omega-3 fatty acid content of from 5% to 50% w/w of the compositionand optionally having one or more the following characteristics orproperties: a) a 2-LPC:1-LPC ratio of from 1:8 to 18:1 or morepreferably from 1:1 to 10:1; b) a phosphatidylcholine (PC) content ofless than 10% w/w of the composition; c) a phosphatidylethanolamine (PE)content of less than 1.2% w/w of the composition; d) a neutral lipidcontent of from 5% to 65% w/w of the composition; e) a 2-LPC ethercontent of less than 1.0% w/w; and f) a ratio of EPA:DHA of from 1:1 to3:1 or a ratio of DHA:EPA of from 1:1 to 5:1.

In some preferred embodiments, the present invention provides marinelysophosphatidylcholine (LPC) compositions or concentrates characterizedin comprising from about 20% to about 40% LPC w/w of the composition andan omega-3 fatty acid content of from 5% to 50% w/w of the compositionand optionally having one or more the following characteristics orproperties: a) a 2-LPC:1-LPC ratio of from 1:8 to 18:1 or morepreferably from 1:1 to 10:1; b) a phosphatidylcholine (PC) content ofless than 10% w/w of the composition; c) a phosphatidylethanolamine (PE)content of less than 1.2% w/w of the composition; d) a neutral lipidcontent of from 5% to 65% w/w of the composition; e) a 2-LPC ethercontent of less than 1.0% w/w; and f) a ratio of EPA:DHA of from 1:1 to3:1 or a ratio of DHA:EPA of from 1:1 to 5:1.

In some preferred embodiments, the present invention provides marinelysophosphatidylcholine (LPC) compositions or concentrates characterizedin comprising from about 40% to about 60% LPC w/w of the composition andan omega-3 fatty acid content of from 5% to 50% w/w of the compositionand optionally having one or more the following characteristics orproperties: a) a 2-LPC:1-LPC ratio of from 1:8 to 18:1 or morepreferably from 1:1 to 10:1; b) a phosphatidylcholine (PC) content ofless than 10% w/w of the composition; c) a phosphatidylethanolamine (PE)content of less than 1.2% w/w of the composition; d) a neutral lipidcontent of from 5% to 65% w/w of the composition; e) a 2-LPC ethercontent of less than 1.0% w/w; and f) a ratio of EPA:DHA of from 1:1 to3:1 or a ratio of DHA:EPA of from 1:1 to 5:1.

In some preferred embodiments, the present invention provides marinelysophosphatidylcholine (LPC) compositions or concentrates characterizedin comprising from about 60% to about 80% LPC w/w of the composition andan omega-3 fatty acid content of from 5% to 50% w/w of the compositionand optionally having one or more the following characteristics orproperties: a) a 2-LPC:1-LPC ratio of from 1:8 to 18:1 or morepreferably from 1:1 to 10:1; b) a phosphatidylcholine (PC) content ofless than 10% w/w of the composition; c) a phosphatidylethanolamine (PE)content of less than 1.2% w/w of the composition; d) a neutral lipidcontent of from 5% to 65% w/w of the composition; e) a 2-LPC ethercontent of less than 1.0% w/w; and f) a ratio of EPA:DHA of from 1:1 to3:1 or a ratio of DHA:EPA of from 1:1 to 5:1.

In some preferred embodiments, the present invention provides marinelysophosphatidylcholine (LPC) compositions or concentrates characterizedin comprising from about 60% to about 100% LPC w/w of the compositionand an omega-3 fatty acid content of from 5% to 50% w/w of thecomposition and optionally having one or more the followingcharacteristics or properties: a) a 2-LPC:1-LPC ratio of from 1:8 to18:1 or more preferably from 1:1 to 10:1; b) a phosphatidylcholine (PC)content of less than 10% w/w of the composition; c) aphosphatidylethanolamine (PE) content of less than 1.2% w/w of thecomposition; d) a neutral lipid content of from 5% to 65% w/w of thecomposition; e) a 2-LPC ether content of less than 1.0% w/w; and f) aratio of EPA:DHA of from 1:1 to 3:1 or a ratio of DHA:EPA of from 1:1 to5:1.

In some embodiments, lysophospholipid compositions described above areprovided for use in increasing the amount of EPA and/or DHA in a targettissue or organ by oral administration of the lysophospholipidcomposition. Preferred target tissues and organ according to theinvention are adrenal gland, blood, bone, bone marrow, brain, fat(white), kidney (whole), large intestine mucosa, liver, lung, muscle,myocardium, pancreas, pituitary gland, prostate gland, skin, smallintestine mucosa, spleen, stomach mucosa, testis, thymus, and/or thyroidgland. In some preferred embodiments, the increase of the amount in EPAand/or DHA is in comparison to an equivalent dose of EPA and/or DHAprovided as a phospholipid (i.e., a phospholipid molecule comprisingfatty acyl groups at both the SN-1 and SN-2 positions of thephospholipid.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: ¹H-NMR spectrum of sample AKB69444-1.

FIG. 2: HPLC chromatogram of LPC product, sample AKB69444-1.

FIG. 3: ¹H-NMR spectrum of sample AKB70005-2.

FIG. 4: HPLC chromatogram of LPC product, sample AKB70005-2.

FIG. 5: ¹H-NMR spectrum of sample AKB70005-3.

FIG. 6: HPLC chromatogram of LPC product of sample AKB70005-3.

FIG. 7: ¹H-NMR spectrum of sample AKB70005-4.

FIG. 8: HPLC chromatogram of LPC product, sample AKB70005-4.

FIG. 9: ¹H-NMR spectrum of sample AKB70005-5.

FIG. 10: HPLC chromatogram of LPC product, sample AKB70005-5.

FIG. 11: Flow chart of processes of the present invention.

FIG. 12: Flow chart of processes of the present invention.

DEFINITIONS

As used herein, “phospholipid” refers to an organic compound that hastwo fatty acid moieties attached at the sn-1 and sn-2 positions ofglycerol and a head group linked by a phosphate residue at the sn-3position of the glycerol. Exemplary headgroup moieties include choline,ethanolamine, serine and inositol. Phospholipids includephosphatidylcholine, phosphatidylserine, phosphatidylethanolamine,phosphatidylinositol and phosphatidic acid. The fatty acid moiety is theportion of the fatty acid molecule that is bound at the sn-1 or sn-2position, for example by an ester or ether linkage. When the fatty acidmoiety is a fatty acyl, the aliphatic chain of the fatty acyl isattached via an ester linkage and when the fatty acid moiety is analiphatic chain of a fatty acid, the aliphatic chain is attached via anether linkage. When a particular fatty acid is mentioned in connectionwith a phospholipid of the invention (e.g., EPA or DHA) it shouldtherefore be taken as a reference to the relevant fatty acyl group or toits aliphatic chain.

As used herein, the term “ether phospholipid” refers to a phospholipidwherein the fatty acid moiety at one of the sn-1 or sn-2 positions is analiphatic chain of a fatty acid attached via an ether linkage. Etherphospholipids include, for example, alkylacylphosphatidylcholine,alkylacylphosphatidylethanolamine and alkylacylphosphatidylserine.

As used herein, the term “lysophospholipid” refers to a phospholipidmolecule that has a fatty acid moiety at one of the sn-1 and sn-2positions of the molecule and an —OH group at the other position so thatthere is one mole of fatty acid moiety per mole of the phospholipidmolecule. The term lysophosphatidylcholine (LPC) refers to aphosphatidylcholine molecule that has a fatty acid moiety at one of thesn-1 and sn-2 positions of the molecule and an —OH group at the otherposition so that there is one mole of fatty acid moiety per mole oflipid. Lysophospholipids may be may be designated as 1- or2-lysophospholipids to denote the position of the —OH group in themolecule. Thus, 1-LPC refers to a lysophosphatidylcholine with an —OHgroup at the sn-1 position of the molecule and a fatty acid moiety atthe sn-2 position. 2-LPC refers to a lysophosphatidylcholine with an —OHgroup at the sn-2 position of the molecule and a fatty acid moiety atthe sn-1 position. When the lysophospholipid is an ether phospholipid,the fatty acid moiety is a fatty alcohol attached via an ether linkageat the sn-1 or sn-2 position. When the lysophospholipid is an esterphospholipid, the fatty acid moiety is a fatty acid ester attached viaan ester linkage at the sn-1 or sn-2 position.

As used herein, the term “long chain polyunsaturated fatty acid” refersto a fatty acid having 20 or more carbons, and which is unsaturated attwo or more bonds.

As used herein, the term omega-3 fatty acid refers to polyunsaturatedfatty acids that have the final double bond in the hydrocarbon chainbetween the third and fourth carbon atoms from the methyl end of themolecule. Non-limiting examples of omega-3 fatty acids include,5,8,11,14,17-eicosapentaenoic acid (EPA),4,7,10,13,16,19-docosahexaenoic acid (DHA) and7,10,13,16,19-docosapentaenoic acid (DPA).

As used herein, the term “physiologically acceptable carrier” refers toany carrier or excipient commonly used with oily pharmaceuticals. Suchcarriers or excipients include, but are not limited to, oils, starch,sucrose and lactose.

As used herein, the term “oral delivery vehicle” refers to any means ofdelivering a pharmaceutical orally, including, but not limited to,capsules, pills, tablets and syrups.

As used herein, the term “food product” refers to any food or feedsuitable for consumption by humans, non-ruminant animals, or ruminantanimals. The “food product” may be a prepared and packaged food (e.g.,mayonnaise, salad dressing, bread, or cheese food) or an animal feed(e.g., extruded and pelleted animal feed or coarse mixed feed).

“Prepared food product” means any pre-packaged food approved for humanconsumption.

As used herein, the term “foodstuff” refers to any substance fit forhuman or animal consumption.

As used herein, the term “functional food” refers to a food product towhich a biologically active supplement has been added.

As used herein, the term “infant food” refers to a food productformulated for an infant such as formula.

As used herein, the term “elderly food” refers to a food productformulated for persons of advanced age.

As used herein, the term “pregnancy food” refers to a food productformulated for pregnant women.

As used herein, the term “nutritional supplement” refers to a foodproduct formulated as a dietary or nutritional supplement to be used aspart of a diet.

As used herein, the term w/w (weight/weight), unless otherwisespecified, refers to the amount of a given substance in a composition ona weight basis and is expressed as a percentage of the total compositionweight. For example, a composition comprising 50% w/w phospholipidsmeans that the mass of the phospholipids is 50% of the total mass of thecomposition (i.e., 50 grams of phospholipids in 100 grams of thecomposition, such as an oil). When solvent concentration is designatedthroughout the specification, the concentration refers to the weightpercent of solvent in the designated solvent solution. As a non-limitingexample, 96% ethanol comprises 96% ethanol and 4% water. As anothernon-limiting example, when the specification describes a lipid solutionas comprising 20% w/w dry matter and that the solvent is 95% ethanol,this means that 100 g of the solution comprises a total of 20 grams drymatter and 80 g of 95% ethanol.

As used herein, the term “krill meal” refers to dried powder preparedfrom krill and having a moisture content of from about 3% to about 15%.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides marine lysophosphatidylcholinecompositions for use in pharmaceuticals, nutraceuticals and functionalfoods, as well as methods for making marine lysophosphatidylcholine(LPC) compositions. In particularly preferred embodiments, the LPCcompositions are prepared from krill phospholipids, for exampleEuphausia superba or Euphausia pacifica phospholipids. In otherpreferred embodiments, the LPC compositions are prepared from Calanus,herring, herring roe, or algal phospholipids. Suitable krill crudephospholipid extracts, desalted krill phospholipid extracts and methodsfor processing krill are disclosed in PCT/IB2016/000208 andPCT/IB2016/000326, each of which is incorporated by reference herein inits entirety.

Intact phospholipids such as phosphatidylcholine are normally convertedto lysophospholipids in the small intestine by the action ofphospholipases. The lysophospholipids are then adsorbed into the body.Data presented herein demonstrate that when LPC compositions areadministered orally in an animal model, that target omega-3 fatty acidsin the composition are incorporated into a large number of tissues atboth a faster rate and in a higher total amount than when orallyadministered in PC (phosphatidylcholine) compositions that do notcontain appreciable amount of lysophospholipids. This result issurprising given the fact that one would expect there to be nodifference because it is known the orally administered PC is convertedin an efficient manner to lysophospholipids in the gut.

1. Starting Materials

The present invention is not limited to the use of any particularbiological starting material. In preferred embodiments, a phospholipidextract is prepared from the biological starting material and the LPCcompositions are made by enzymatic treatment of the phospholipidextract. The biological starting material may preferably be or beproduced from an algal biomass, plant biomass or marine animal biomass.In preferred embodiments, marine animal biomasses are utilized as thestarting material. Suitable marine animal biomasses include, but are notlimited to krill, crabs, Calanus, plankton, eggs, crayfish, shrimp,fish, especially herring, and molluscs. The biological starting materialcan be either fresh or frozen, or can be a material produced from analgal, plant or marine animal biomass such as a meal, powder,hydrolysate, or coagulate (paste). The paste may be a wet paste or adried paste. In some preferred embodiments, the biological startingmaterial is a krill material, for example a krill meal, krillhydrolysate, krill coagulate, or fresh or frozen krill. Any species ofkrill may be utilized. In preferred embodiments, the krill is Euphausiasuperba or Euphausia pacifica.

In some particularly preferred embodiments, the biological startingmaterial is a krill meal. Krill meal can be preferably be made by anystandard marine meal process. In general, the krill meal is produced bycooking freshly caught krill at low temperature (approximately 80-85°C.) and drying to reduce the moisture content to approximately 5 to 8%and then grinding. In embodiments where the product is intended forhuman consumption, it is preferable to pack and store the meal undernitrogen without the addition of antioxidants.

Accordingly, the processes of the present invention may be used with awide variety of starting materials. The remainder of the discussion ofthe processes generally refer to the use of krill meal as the startingmaterial. However, it will be understood that any of the startingmaterials considered herein may be substituted for krill meal in thedescribed processes.

2. Solvent Extraction from Krill Meal

In the first step of the extraction process, the krill meal is mixedwith a suitable solvent to extract lipids from the meal. In contrast toprior art methods, the present invention utilizes conditions whichpreferably extract the maximum amount of lipids from the krill meal atthe cost of an increased amount of contaminants in the initial solventextract. In preferred embodiments, the solvent is an organic proticsolvent, however other solvents known for use in extraction of foodgrade lipids may also be used such as acetone, hexane, etc. Suitableorganic protic solvents include, but are not limited to, n-butanol,n-propanol, isopropanol, nitromethane, ethanol, and methanol. Inparticularly preferred embodiments, the protic solvent is ethanol.

In preferred embodiments, the concentration of the protic solvent usedin the initial solvent extraction step is at least 90%, or preferablyfrom about 94% to 98%, more preferably from about 95% to 97%, and ismost preferably about 96% (e.g., 96% ethanol or methanol).

In some embodiments, the protic solvent is mixed with the biologicalstarting material at a ratio of protic solvent:biological startingmaterial of about 1:1 to 10:1, preferably about 3:1 to 6:1, morepreferably about 4:1 to 5:1, and most preferably about 4.4:1.

In preferred embodiments, the biological starting material is extractedwith protic solvent at a temperature of from about 5° C. to 65° C., fromabout 20° C. to about 60° C., preferably from about 30° C. to 50° C.,more preferably from about 30° C. to 50° C., and most preferably atabout 40° C. In some embodiments, the extraction time (i.e., the amountof time the biological starting material is in contact with the solvent)is from about 10 minutes to about 2 hours, preferably from about 15minutes to 60 minutes, more preferably from about 20 minutes to about 45minutes, and most preferably about 30 minutes.

Following the extraction step, a crude krill lipid solution containingthe lipids from the krill meal is separated from the solvent/krill mealmixture, for example by decantation and or filtration. The insolublematerial, comprising proteins and other useful materials is then driedto recover ethanol. The remaining protein-rich meal product maysubsequently be used in food products, protein supplements, animal feedsand the like. In some embodiments, the decanted solution containingsoluble lipids has a dry matter content of from about 4% to 9% w/w,preferably from about 5.5% to 7.5% w/w, and most preferably from about6% to 7% w/w, where w/w refers to the weight of dry matter as a percentof the total solution weight. In preferred embodiments, the dry matterconsists essentially of crude krill lipids, and preferably has a lipidcontent of greater than 90%, 95%, 96%, 97%, 98% or 99% w/w, wherein w/wrefers to the weight of lipids a percent of the total dry matter weight.

3. Desalting and Concentration

In some embodiments, the crude krill lipid solution is desalted toremove hexane insoluble materials such as insoluble inorganic salts(e.g., NaCl with small or trace amounts of KCl and/or AlCl₃) as well asunwanted compounds such as trimethylamine oxide, and metals such ascopper and arsenic. In some preferred embodiments, the LPC compositionof the present invention is prepared from the desalted krill lipidcomposition. While the methods are described in reference to thedesalted krill lipids described above, the methods are generallyapplicable any lipid fractions that contain phospholipids.

In some embodiments, the crude krill lipid solution is desalted byevaporating the solvent from crude krill lipid solution to provide acrude krill lipid composition and then subjecting the crude krill lipidcomposition to repeated washes with an aqueous solvent. Suitable aqueoussolvents include, but are not limited to, ethanol blended with water ordeionized water so that the ethanol concentration is from about 40% to70%, preferably about 50% to 60%. In these embodiments, the crude krilllipid composition is mixed with the solvent, the lipid phase isrecovered, and the aqueous phase is decanted. The washing step may berepeated as needed, for example 1, 2, 3, 4, 5 or more times. The rationof aqueous solvent:crude krill lipid composition is preferably fromabout 1:1 to 1:5 for each wash step, more preferably from about 1:1 to2.5:1, and most preferably about 1:1.7.

In some embodiments, the crude lipid solution is desalted bychromatography. Suitable chromatographic media include silica gel media,including but not limited to spherical silica gels and derivatizedsilica gels such as C8 (octyl functionalized silica) and C18 (octadecylfunctional silica) and ion exchange resins such as Dowex™ resins. Inembodiments where chromatography is utilized, the crude krill lipids arepreferably applied to the chromatographic medium in a protic solvent,preferably the same solvent used in the initial extraction (e.g.,ethanol). Standard column chromatography methods may be utilized,however, moving bed chromatography or simulated moving bedchromatography apparatuses may preferably be utilized. The presentinvention is not limited to any particular type of chromatographicpurification apparatus. Indeed, in preferred embodiments, thechromatographic purification apparatus may be a column, a fixed bedapparatus, a simulated moving bed apparatus or a moving bed apparatus.In some particularly preferred embodiments, the apparatus is a simulatedmoving bed (SMB) apparatus. Suitable SMB systems are disclosed, forexample, in U.S. Pat. Nos. 9,556,116; 9,650,590; and 9,560,333; each ofwhich is incorporated herein by reference in its entirety.

The composition of the desalted krill lipids on a dry matter basis maybe preferably characterized as follows. In some embodiments, thedesalted krill lipids preferably comprise from about 30% w/w to 50% w/wphospholipids, more preferably from about 35% w/w to about 45% w/wphospholipids, and most preferably about 40% w/w phospholipids, whereinw/w refers to the weight of the phospholipids as a percent of the totaldesalted kill lipid weight. In some embodiments, the desalted krilllipids preferably comprise from about 32% w/w to 52% w/w triglycerides,more preferably from about 36% w/w to about 48% w/w triglycerides, andmost preferably about 42% w/w triglycerides, wherein w/w refers to theweight of the triglycerides as a percent of the total desalted krilllipid weight. In some embodiments, the desalted krill lipids preferablycomprise from about 3% w/w to 13% w/w free fatty acids, more preferablyfrom about 5% w/w to about 11% w/w free fatty acids, and most preferablyabout 8% w/w free fatty acids, wherein w/w refers to the weight of thefree fatty acids as a percent of the total desalted krill lipid weight.In some embodiments, the desalted krill lipids preferably comprise fromabout 0.5% w/w to 5% w/w lysophospholipids, more preferably from about0.8% w/w to about 3.2% w/w lysophospholipids, and most preferably about1.2% to 2.8% w/w lysophospholipids, wherein w/w refers to the weight ofthe lysophospholipids as a percent of the total desalted krill lipidweight. In some embodiments, the desalted krill lipids preferablycomprise less than about 1% w/w inorganic salts, more preferably lessthan about 0.5% w/w inorganic salts, even more preferably less thanabout 0.2% w/w w/w inorganic salts, and most preferably less than about0.1% w/w inorganic salts, wherein w/w refers to the weight of theinorganic salts as a percent of the total desalted krill lipid weight.In some embodiments, the desalted krill lipids preferably comprise lessthan about 5 mg N/100 g, more preferably less than about 3 mg N/100 g,even more preferably less than about 2 mg N/100 g, and most preferablyless than about 1 mg N/100 g, where the N content serves as a convenientproxy for trimethylamine oxide (TMAO) content. In some embodiments, thedesalted krill lipids comprise less than about 10 ppm copper (Cu⁺⁺),more preferably less than about 5 ppm Cu⁺⁺, even more preferably lessthan about 2 ppm Cu⁺⁺, and most preferably less than about 1 ppm Cu⁺⁺.In some embodiments, the desalted krill lipids comprise less than about10 ppm total arsenic (As³⁺, organic and inorganic), more preferably lessthan about 5 ppm total arsenic, even more preferably less than about 3ppm total arsenic, and most preferably less than about 1 ppm totalarsenic. In some embodiments, the desalted krill lipids preferablycomprise from about 0.01% to 2% w/w ethyl esters, more preferably fromabout 0.01% to about 1.5% w/w ethyl esters, and most preferably fromabout 0.01% to about 1% w/w ethyl esters, wherein w/w refers to theweight of the ethyl esters as a percent of the total desalted krilllipid weight. In some embodiments, the krill phospholipid compositionspreferably comprise less than about 5%, 4%, 3% or 2% w/w ethyl estersdown to a lower limit of 0.01% ethyl esters (i.e., between 5% and 0.01%w/w ethyl esters, between 4% and 0.01% w/w ethyl esters, between 3% and0.01% w/w ethyl esters, or between 2% and 0.01% w/w ethyl esters), morepreferably less than about 1.5% w/w ethyl esters, and most preferablyless than about 1% w/w ethyl esters, wherein w/w refers to the weight ofthe ethyl esters as a percent of the total desalted krill lipid weight.In some embodiments, the desalted krill lipids have a conductivity ofless than about 50 μS/cm when measured with 5% dry matter in 95%ethanol, more preferably a conductivity of less than about 30 μS/cm whenmeasured with 5% dry matter in 95% ethanol, and most preferably aconductivity of less than about 20 μS/cm when measured with 5% drymatter in 95% ethanol. In some embodiments, the desalted krill lipidshave a viscosity of from about 50 to 800 mPas at 25° C., more preferablyfrom about 100 to 400 mPas at 25° C., and most preferably 180 to 340mPas at 25° C. In some embodiments, the desalted krill lipidcompositions have a pH of from about 6.7 to 8.3 when measured in 95%ethanol.

In some preferred embodiments, the LPC compositions of the presentinvention are prepared from a phospholipid composition made from thedesalted krill lipid composition. While the methods are described inreference to the desalted krill lipids described above, the methods aregenerally applicable any lipid fractions that contain phospholipids.

Accordingly, in some embodiments, the dry matter content of a lipidcomposition containing phospholipids, such as the desalted krill lipidcomposition described above, is adjusted to a predetermined level byadding or removing solvent and the resulting mixture is allowed tofractionate so that the phospholipids are predominantly partitioned intoone phase and the neutral lipids partitioned into a different phase. Insome embodiments, a lipid composition containing phospholipids such asthe desalted krill lipids is mixed with a suitable protic solvent,preferably ethanol, so that the dry matter (i.e., lipid) content of theresulting solution is from about 10% to 40% w/w, preferably about 15% to35% w/w, more preferably about 18% to 30% w/w, and most preferably about20% to 25% w/w. In embodiments where the desalting step already providesthe lipids in a suitable protic alcohol solution, such as is the casewhere ethanol is used as the solvent for chromatography, the desaltedkrill lipid solution may preferably be evaporated to provide desired drymatter content, i.e., from about 10% to 40% w/w, preferably about 15% to35% w/w, more preferably about 18% to 28% w/w, and most preferably about20% to 22% w/w. Suitable methods for evaporation include, but are notlimited to, evaporation under reduced pressure (e.g., vacuumdistillation), falling film evaporation, and removal of solvents via amembrane.

Following adjustment of the dry matter content to the desired level byeither adding or removing solvent, the solution is then allowed tofractionate into an upper, light phase solution with an enrichedphospholipid content and a lower, heavy phase solution containingpredominantly neutral lipids and a high level of astaxanthin.Preferably, the temperature of the solution during the fractionationstep is controlled. In some embodiments, the temperature for thefractionation step is from about 0° C. to about 20° C., preferably fromabout 5° C. to about 15° C., more preferably from about 8° C. to about12° C., and most preferably about 10° C.

In some embodiments, the concentration of the protic solvent may bevaried in order to control the phospholipid concentration in the lipidcomposition of the upper phase. In some embodiments, the protic solventhas a concentration of from about 55% to 100%, more preferably about 65%to 98%. In some preferred embodiments, the protic solvent has aconcentration of from about 90% to 100%, more preferably about 92% to98%, and most preferably about 95%. In these embodiments, thephospholipid content on a dry matter basis of the lipids in the upperphase after fractionation is from about 50% to 70% w/w, preferably about55% to 65% w/w and most preferably about 60% w/w. In still otherpreferred embodiments, the protic solvent has a concentration of fromabout 80% to 90% w/w, more preferably about 82% to 88% w/w, and mostpreferably about 85% w/w. In these embodiments, the phospholipid contenton a dry matter basis of the lipids in the upper phase afterfractionation is from about 70% to 90% w/w, preferably about 75% to 85%w/w and most preferably about 80% w/w.

In some embodiments, the upper and lower phases are separated bycentrifugation, preferably cryocentrifugation with a two phase or threephase separator. In some embodiments, the centrifugation is conducted atfrom about 0° C. to about 30° C., more preferably from about 0° C. toabout 10° C. and most preferably from about 3° C. to about 7° C. Ingeneral, the gravitational force utilized will depend on delta T betweenthe phases. Lower temperatures provide a greater delta T. In somepreferred embodiments, the G force employed in the separation is fromabout 8000×G to about 15000×G.

In some embodiments, the process steps of adjusting the dry mattercontent as described above through the centrifugation steps are repeatedone or more times.

In some embodiments, the upper light phase is collected and processedfurther. The solvent is preferably removed from the upper phase by oneor more evaporation steps to yield a krill phospholipid composition. Thekrill phospholipid compositions preferably comprise from about 50% to85% w/w phospholipids, and more preferably from about 55% to 80% w/wphospholipids, wherein w/w refers to the weight of phospholipids as apercent of the total weight of the composition.

In some embodiments, the lower heavy phase is collected and processedfurther. In some embodiments, the solvent is removed from the lowerphase to provide a krill neutral lipid composition. In some embodiments,the lower phase may be fractionated with protic solvent and subjected toa second centrifugation step to recover additional phospholipids notrecovered in the first fractionation step. Again, the solvent is removedfrom the resulting lower phase to provide a krill neutral lipidcomposition. The krill neutral lipid composition in both instances incharacterized in containing high levels of astaxanthin. In someembodiments, the krill neutral lipid composition may be combined orblended with the krill phospholipid composition to provide a lipidcomposition with desired levels of phospholipids, neutral lipids, andastaxanthin. In some embodiments, the krill neutral lipid compositionmay be further processed (e.g., by chromatography) to provide anastaxanthin composition. The astaxanthin composition may then becombined or blended with the krill phospholipid composition to provide alipid composition with desired levels of phospholipids and astaxanthin.

In some embodiments, the processes further comprise the step of adding atriglyceride oil, such as medium chain triglyceride oil or long chaintriglyceride oil, at any stage during the process. For example, thetriglyceride oil may be added to the collected light phase, heavy phase,phospholipid composition or neutral lipid composition. In someembodiments, the process steps of adjusting the dry matter content asdescribed above through the centrifugation steps and/or evaporationsteps are repeated one or more times.

In some embodiments, krill phospholipid and neutral lipid compositionsare produced by a further chromatography step. In these embodiments, atleast a portion of the desalted lipid rich stream described above isintroduced into to a polar liquid extraction zone comprising a fixed bedadsorber containing a macroporous styrenic polymeric bead type resineffective to adsorb neutral lipids. In preferred embodiments, the fixedbed chromatography step provides a polar lipid extract stream comprisingsolvent and at least 50 wt-% polar lipids on a dry basis. In somepreferred embodiments, the fixed bed adsorber is intermittentlyregenerated with a hot ethanol stream at a hot regeneration temperaturebetween about 40° C. and about 60° C. to provide a neutral lipidraffinate stream comprising solvent, neutral lipids and astaxanthin. Insome embodiments, solvent is recovered polar lipid extract stream toprovide a krill phospholipid composition and from the neutral lipidraffinate stream to provide a neutral lipid composition. These processesare described in more detail in co-pending U.S. application Ser. No.14/619,102, which is incorporated herein by reference in its entirety.

In some embodiments, the krill phospholipid compositions on a dry matterbasis preferably comprise from about 5% w/w to 35% w/w triglycerides,more preferably from about 10% w/w to about 30% w/w triglycerides, andmost preferably about 15% to 25% w/w triglycerides, wherein w/w refersto the weight of the triglycerides as a percent of the total krillphospholipid composition weight. In some embodiments, the krillphospholipid compositions preferably comprise from about 2% w/w to 13%w/w free fatty acids, more preferably from about 4% w/w to about 11% w/wfree fatty acids, and most preferably about 4% to 10% w/w free fattyacids, wherein w/w refers to the weight of the free fatty acids as apercent of the total krill phospholipid composition weight. In someembodiments, the krill phospholipid compositions preferably comprisefrom about 0.5% w/w to 10% w/w lysophospholipids, more preferably fromabout 0.8% w/w to about 7.0% w/w lysophospholipids, and most preferablyless than about 5.0% w/w or 3.0% w/w lysophospholipids, wherein w/wrefers to the weight of the lysophospholipids as a percent of the totalkrill phospholipid composition weight. In some embodiments, the krillphospholipid compositions preferably comprise less than about 1% w/winorganic salts, more preferably less than about 0.5% w/w inorganicsalts, even more preferably less than about 0.2% w/w inorganic salts,and most preferably less than about 0.1% or 0.05% w/w inorganic salts,wherein w/w refers to the weight of the inorganic salts as a percent ofthe total krill phospholipid composition weight. In some embodiments,the krill phospholipid composition preferably comprises less than about5 mg N/100 g, more preferably less than about 3 mg N/100 g, even morepreferably less than about 2 mg N/100 g, and most preferably less thanabout 1 mg N/100 g, where the N content serves as a convenient proxy fortrimethylamine oxide (TMAO) content. In some embodiments, the krillphospholipid compositions comprise less than about 10 ppm copper (Cu⁺⁺),more preferably less than about 5 ppm Cu⁺⁺, even more preferably lessthan about 2 ppm Cu⁺⁺, and most preferably less than about 1 ppm Cu⁺⁺.In some embodiments, the krill phospholipid compositions comprise lessthan about 10 ppm total arsenic (As³⁺), more preferably less than about5 ppm total arsenic, even more preferably less than about 3 ppm totalarsenic, and most preferably less than about 1 ppm total arsenic. Insome embodiments, the krill phospholipid composition preferably comprisefrom about 0.01% to 2% w/w ethyl esters, more preferably from about0.01% to about 1.5% w/w ethyl esters, and most preferably from about0.01% to about 1% w/w ethyl esters, wherein w/w refers to the weight ofthe ethyl esters as a percent of the total krill phospholipidcomposition weight. In some embodiments, the krill phospholipidcomposition preferably comprise less than about 5%, 4%, 3% or 2% w/wethyl esters down to a lower limit of 0.01% ethyl esters (i.e., between5% and 0.01% w/w ethyl esters, between 4% and 0.01% w/w ethyl esters,between 3% and 0.01% w/w ethyl esters, or between 2% and 0.01% w/w ethylesters), more preferably less than about 1.5% w/w ethyl esters, and mostpreferably less than about 1% w/w ethyl esters, wherein w/w refers tothe weight of the ethyl esters as a percent of the total krillphospholipid composition weight. In some embodiments, the krillphospholipid composition have a conductivity of less than about 50 μS/cmwhen measured with 5% dry matter in 95% ethanol, more preferably aconductivity of less than about 30 μS/cm when measured with 5% drymatter in 95% ethanol, and most preferably a conductivity of less thanabout 20 μS/cm, 10 μS/cm, 5 μS/cm or 1 μS/cm when measured with 5% drymatter in 95% ethanol. In some embodiments, the krill phospholipidcomposition has a viscosity of from about 400 to 2000 mPas at 35° C.,more preferably from about 500 to 1800 mPas at 35° C., and mostpreferably from about 600 to 1600 mPas at 35° C. In some embodiments,the krill phospholipid composition has a pH of from about 6.7 to 8.3when measured in 95% ethanol.

4. Production of LPC Composition

In some preferred embodiments, the LPC compositions of the presentinvention are prepared from the phospholipid sources described above. Insome preferred embodiments, the present invention provides methods formaking a lysophosphatidylcholine (LPC) composition with a high contentof EPA and DHA from a marine or other raw material containingphospholipids comprising treating the marine raw material with aphospholipase that is not native to the marine raw material to provide aphospholipase treated raw material and fractionating the phospholipasetreated raw material to provide a lipid composition having a higherlysophosphatidylcholine content than the starting raw material. In somepreferred embodiments, the raw material is selected from the groupconsisting of a krill lipid preparation, a herring lipid preparation, aherring roe lipid preparation, an algal lipid preparation, and a Calanuslipid preparation, thereby providing a krill LPC composition, a herringLPC composition, a herring roe LPC composition, an algal LPCcomposition, or a Calanus LPC composition. In some particularlypreferred embodiments, the krill lipid preparation is a Euphausiasuperba lipid preparation. In some preferred embodiments, the rawmaterial has a content of EPA and DHA at the range of; EPA: 1-70 wt %and DHA: 1-70 wt %, more preferably EPA: 5-70 wt % and DHA: 5-70 wt %,most preferably EPA: 10-60 wt % and DHA: 10-60 wt %.

In some embodiments, the raw material is contacted with a phospholipasein a solvent. The present invention is not limited to the use of anyparticular phospholipase. In some embodiments, the phospholipase is aphospholipase A1 (PLA1). In some particularly preferred embodiments, theenzyme is LECITASE™ Ultra, QUARA™ LowP. In some embodiments, the solventis a mixture of water and an alcohol. In some preferred embodiments, thealcohol is ethanol. In still further preferred embodiments, the rawmaterial is contacted with a phospholipase in a mixture of about 85%water and 15% ethanol. In some particularly preferred embodiments, theenzyme is a phospholipase A1 (PLA1), and the enzyme concentration is inthe range of 0.1-20 vol/wt %, preferably 0.1-15 vol/wt %, morepreferably 0.1-10 vol/wt %, further preferably 0.1-5 vol/wt %, mostpreferably 0.1-3 vol/wt %. In some preferred embodiments, the enzyme isa phospholipase A1 (PLA1), wherein the method is carried out at a pH of3-12, preferably 4-10, more preferably 4-9, most preferably 5-9. In somepreferred embodiments, the enzyme is a phospholipase A1 (PLA1), whereinthe method is carried out between 4-95° C., preferably 4-85° C., morepreferably 10-80° C., further preferably 15-70° C., even more preferably15-65° C., most preferably 15-60° C.

In some preferred embodiments, the LPC compositions of the presentinvention are prepared by additional solvent concentration and/orchromatographic separation procedures. In some preferred embodiments,the enzyme-treated LPC composition is concentrated by phase separationin a polar/non-polar solvent system. In some preferred embodiments, theenzyme-treated LPC composition is mixed with a solvent system comprisingequal amounts of a polar solvent (e.g., methanol) and a non-polarsolvent (e.g., heptane). The resulting mixture is agitated, and thephases allowed to separate. The polar lipids (e.g., LPC and PC)partition into the polar phase. In some embodiments, the polar phase isremoved, washed with heptane, and the polar phase is recovered. In somepreferred embodiments, the polar phase is then evaporated to dryness toprovide a concentrated LPC composition. In some embodiments, the LPCcomposition may be further concentrated by chromatographic procedures,for example, by flash chromatography using silica 60 gel followed byevaporation to dryness.

The methods described above produce LPC compositions with preferredcharacteristics and/or properties. In some preferred embodiments, theLPC composition has a content of EPA and DHA in the range of; EPA: 1-100wt % and/or DHA: 1-100 wt %, more preferably EPA: 5-100 wt % and/or DHA:5-100 wt %, most preferably EPA: 10-90 wt % and/or DHA: 10-90 wt %. Instill further preferred embodiments, the LPC composition has an LPCcontent in the ranges of 10-100 wt %, preferably 20-100 wt %, morepreferably 30-100 wt %, further preferably 40-100 wt %, most preferably50-100 wt %.

In still other preferred embodiments, the LPC composition obtained bythe process is characterized in comprising:

about 10% to about 100% LPC w/w of the composition, or from 60% to 100%LPC, from 70% to 90% LPC, from 20% to 50% LPC, from 20% to 40% LPC, from20% to 30% LPC, 10% to 30% LPC and by having an omega-3 fatty acidcontent of from 5% to 60% w/w of the composition, from 5% to 50% w/w ofthe composition, from 5% to 40% of the composition or from 5% to 30% ofthe composition, or from 20% to 60% w/w of the composition, from 20% to50% w/w of the composition, or from 20% to 40% w/w of the composition,or from 30% to 50% w/w of the composition. It will be recognized bythose of skill in the art that the omega-3 fatty acid content includesthe content of omega-3 fatty acid acyl chains that are linked by esteror ether bonds to phospholipid and glycerol molecules in thecomposition. The wt % of omega-s fatty acid acyl groups in thecomposition may preferably be determined by ¹H-NMR or other suitable NMRtechniques, including ¹³C-NMR. Alternatively, the omega-3 fatty acidcontent may be expressed in g/100 g of the fatty acids as analyzed bygas chromatography as is known in the art. In these embodiments, thelysophospholipid compositions preferably comprise from 10 to 50 g/100 gomega-3 fatty acids and most preferably from 20 to 40 g/100 g omega-3fatty acids where g/100 grams is the weight of the omega-3 fatty acidsper 100 grams of total fatty acids as measured by gas chromatography.

In preferred embodiments, the lysophospholipid compositions additionallyhave one or more the following characteristics or properties:

a) a 2-LPC:1-LPC ratio of from 1:8 to 18:1, and more preferably from1.2:1 to 8:1, 1.2:1 to 4:1, 1.2:1 to 2:1, 4:1 to 10:1; or 5:1 to 12:1;

b) a phosphatidylcholine (PC) content of from 0.5% to 10% w/w of thecomposition, and more preferably less than 10%, 9%, 8%, 7%, 6% or 5% w/wof the composition;

c) a phosphatidylethanolamine (PE) content of less than 1.2%, 1.1%,1.0%, 0.7% or 0.5% w/w of the composition;

d) a neutral lipid content of from about 5% to 65% w/w of thecomposition, or more preferably from about 45% to 65% w/w or 15% to 35%w/w;

e) a ratio of 2-LPC:2-LPC ether of from 15:1 to 50:1, and morepreferably from 50:1 to 25:1 and wherein the compositions can preferablycomprise less than 5%, 4%, 3%, 2%, 1.0%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%,0.4%, 0.3%, 0.2% or 0.1% 2-LPC-ether; and/or

f) a ratio of EPA:DHA of from 1:1 to 3:1 of DHA:EPA of from 1:1 to 5:1.

In some preferred embodiments, the compositions from comprise from 20%to 40% or from 23% to 35% LPC w/w of the composition. In some preferredembodiments, the compositions comprise from 40% to 60%, from 40% to 70%,or from 50% to 70% LPC w/w of the composition. In some preferredembodiments, the composition comprises from 70% to 90% LPC w/w of thecomposition. With regard to property (f), where the LPC composition isprepared from krill phospholipids, the LPC composition will preferablycomprise a ratio of EPA:DHA of from 1:1 to 3:1 and more preferably from1.5:1 to 2:5:1. With further regard to property (f), where the LPCcomposition is prepared from marine sources other than krill (e.g.,herring, herring roe or marine algae), the LPC composition willpreferably comprise a ratio of DHA:EPA of from 1:1 to 5:1, morepreferably from 2:1 to 4:1, and most preferably from 2.5:1 to 3.5:1.

In some preferred embodiments, the composition has an omega-3 fatty acidcontent of from 35% to 45% w/w of the composition. In some preferredembodiments, the composition has property (a), (b), (c), (d), (e) or (f)or a combination the properties, for example: properties (a) and (b);(a) and (c); (a) and (d); (a) and (e); (a) and (f) (b) and (c); (b) and(d); (b) and (e); (b) and (f); (c) and (d); (c) and (e); ((c) and (f);(d) and (e); (d) and (f); (e) and (f); (a), (b) and (c); (a), (b) and(d); (a), (b) and (e); (a), (b) and (f); (a), (c) and (d); (a), (c) and(e); (a), (c) and (f); (a), (d) and (e); (a), (d) and (f); (b), (c) and(d); (b), (c) and (e); (b) (c) and) f); (b), (d) and (e); (b), (d) and(f); (c), (d) and (e); (c), (d) and (f); (a), (b), (c), and (d); (a),(b), (c), and (e); (a), (b), (c) and (f); (b), (c), (d), and (e); (b),(c), (d) and (f); (a), (c), (d), and (e); (a), (c), (d) and (f); (a),(b), (d), and (e); (a), (b), (d) and (f); (a), (b), (c), (d) and (e);(a), (b), (c), (d), and (f); (b), (c), (d), (e), and (f); (a), (b), (d),(e) and (f); (a), (b), (c), (e) and (f); and (a), (b), (c), (d), (e) and(f) as well as any other possible combinations. In some preferredembodiments, the compositions have two or more of properties (a), (b)and (c). In some preferred embodiments, the composition has two or moreof properties (a), (b), (c), (d) and (e). In some preferred embodiments,the composition has three or more of properties (a), (b), (c), (d), (e)and (f). In some preferred embodiments, the composition has four or moreof properties (a), (b), (c), (d) and (e). In some preferred embodiments,the composition has five or more of properties (a), (b), (c), (d), (e)and (f).

5. Formulation of LPC Compositions

In some preferred embodiments, the present invention provides apharmaceutical or nutraceutical composition comprising an LPCcomposition as described above and a physiologically acceptable carrier.In some preferred embodiments, the physiologically acceptable carrier isa lipid carrier. In some preferred embodiments, the present inventionprovides an oral delivery vehicle containing a marine LPC composition,pharmaceutical composition or nutraceutical composition as describedherein.

In some preferred embodiments, the present invention provides a lipidcomposition comprising a lipid fraction and second lipid fraction,wherein the first lipid fraction is the marine LPC composition asdescribed herein and the second lipid fraction is obtained from adifferent source than the first lipid fraction and/or contains less than20% LPC. In some preferred embodiments, the second lipid fraction isselected from the group consisting of a triglyceride fraction, adiglyceride fraction, a fatty acid ethyl ester fraction, a free fattyacid fraction and combinations thereof. In some preferred embodiments,the second lipid fraction is a marine lipid fraction comprising EPAand/or DHA. In some preferred embodiments, the present inventionprovides a pharmaceutical or nutraceutical composition comprising thelipid composition just described and a physiologically acceptablecarrier. In some preferred embodiments, the present invention providesan oral delivery vehicle containing the lipid compositions justdescribed.

The LPC compositions of the present invention are preferablyadministered orally. Accordingly, in some embodiments, the compositionsof this invention (such as those described in the preceding sections)are contained in acceptable excipients and/or carriers for oralconsumption. The actual form of the carrier, and thus, the compositionitself, is not critical. The carrier may be a liquid, gel, gelcap,capsule, powder, solid tablet (coated or non-coated), tea, or the like.The composition is preferably in the form of a tablet or capsule andmost preferably in the form of a soft gel capsule. Suitable excipientand/or carriers include vegetable oil, fish oil, krill oil,maltodextrin, calcium carbonate, dicalcium phosphate, tricalciumphosphate, microcrystalline cellulose, dextrose, rice flour, magnesiumstearate, stearic acid, croscarmellose sodium, sodium starch glycolate,crospovidone, sucrose, vegetable gums, lactose, methylcellulose,povidone, carboxymethylcellulose, corn starch, and the like (includingmixtures thereof). Preferred carriers include calcium carbonate,magnesium stearate, maltodextrin, and mixtures thereof. The variousingredients and the excipient and/or carrier are mixed and formed intothe desired form using conventional techniques. The tablet or capsule ofthe present invention may be coated with an enteric coating thatdissolves at a pH of about 6.0 to 7.0. A suitable enteric coating thatdissolves in the small intestine but not in the stomach is celluloseacetate phthalate. Further details on techniques for formulation for andadministration may be found in the latest edition of Remington'sPharmaceutical Sciences (Maack Publishing Co., Easton, Pa.).

In some embodiments, the LPC compositions are formulated for oraladministration with flavoring agents or sweeteners. Examples of usefulflavoring include, but are not limited to, pure anise extract, imitationbanana extract, imitation cherry extract, chocolate extract, pure lemonextract, pure orange extract, pure peppermint extract, imitationpineapple extract, imitation rum extract, imitation strawberry extract,or pure vanilla extract; or volatile oils, such as balm oil, bay oil,bergamot oil, cedarwood oil, walnut oil, cherry oil, cinnamon oil, cloveoil, or peppermint oil; peanut butter, chocolate flavoring, vanillacookie crumb, butterscotch or toffee. In one embodiment, the dietarysupplement contains cocoa or chocolate. Emulsifiers may be added forstability of the final product. Examples of suitable emulsifiersinclude, but are not limited to, lecithin (e.g., from egg or soy),and/or mono- and di-glycerides. Other emulsifiers are readily apparentto the skilled artisan and selection of suitable emulsifier(s) willdepend, in part, upon the formulation and final product. In addition tothe carbohydrates described above, the nutritional supplement cancontain natural or artificial (preferably low calorie) sweeteners, e.g.,saccharides, cyclamates, aspartamine, aspartame, acesulfame K, and/orsorbitol.

The LPC compositions of the present invention may also be delivered asdietary supplements, nutritional supplements, or functional foods.

The dietary supplement may comprise one or more inert ingredients,especially if it is desirable to limit the number of calories added tothe diet by the dietary supplement. For example, the dietary supplementof the present invention may also contain optional ingredientsincluding, for example, herbs, vitamins, minerals, enhancers, colorants,sweeteners, flavorants, inert ingredients, and the like. For example,the dietary supplement of the present invention may contain one or moreof the following: ascorbates (ascorbic acid, mineral ascorbate salts,rose hips, acerola, and the like), dehydroepiandosterone (DHEA), greentea (polyphenols), inositol, kelp, dulse, bioflavonoids, maltodextrin,nettles, niacin, niacinamide, rosemary, selenium, silica (silicondioxide, silica gel, horsetail, shavegrass, and the like), Spirulina,zinc, and the like. Such optional ingredients may be either naturallyoccurring or concentrated forms.

In some embodiments, the dietary supplements further comprise vitaminsand minerals including, but not limited to, calcium phosphate oracetate, tribasic; potassium phosphate, dibasic; magnesium sulfate oroxide; salt (sodium chloride); potassium chloride or acetate; ascorbicacid; ferric orthophosphate; niacinamide; zinc sulfate or oxide; calciumpantothenate; copper gluconate; riboflavin; beta-carotene; pyridoxinehydrochloride; thiamin mononitrate; folic acid; biotin; chromiumchloride or picolonate; potassium iodide; sodium selenate; sodiummolybdate; phylloquinone; vitamin D₃; cyanocobalamin; sodium selenite;copper sulfate; vitamin A; vitamin C; inositol; potassium iodide.Suitable dosages for vitamins and minerals may be obtained, for example,by consulting the U.S. RDA guidelines.

In other embodiments, the present invention provides nutritionalsupplements (e.g., energy bars or meal replacement bars or beverages)comprising of the LPC compositions of the present invention. Inpreferred embodiments, the nutritional supplements comprise an effectiveamount of the components as described above. The nutritional supplementmay serve as meal or snack replacement and generally provide nutrientcalories. Preferably, the nutritional supplements provide carbohydrates,proteins, and fats in balanced amounts. The nutritional supplement canfurther comprise carbohydrate, simple, medium chain length, orpolysaccharides, or a combination thereof. A simple sugar can be chosenfor desirable organoleptic properties. Uncooked cornstarch is oneexample of a complex carbohydrate. If it is desired that it shouldmaintain its high molecular weight structure, it should be included onlyin food formulations or portions thereof which are not cooked or heatprocessed since the heat will break down the complex carbohydrate intosimple carbohydrates, wherein simple carbohydrates are mono- ordisaccharides. The nutritional supplement contains, in one embodiment,combinations of sources of carbohydrate of three levels of chain length(simple, medium and complex; e.g., sucrose, maltodextrins, and uncookedcornstarch).

In still further embodiments, the present invention provides foodproducts, prepared food products, or foodstuffs (i.e., functional foods)comprising the LPC compositions of the present invention. In preferredembodiments, the foods comprise an effective amount of the components asdescribed above. For example, in some embodiments, beverages and solidor semi-solid foods comprising the fatty acids or derivatives thereofare provided. These forms can include, but are not limited to, beverages(e.g., soft drinks, milk and other dairy drinks, and diet drinks), bakedgoods, puddings, dairy products, confections, snack foods, or frozenconfections or novelties (e.g., ice cream, milk shakes), prepared frozenmeals, candy, snack products (e.g., chips), soups, spreads, sauces,salad dressings, prepared meat products, cheese, yogurt and any otherfat or oil containing foods, and food ingredients (e.g., wheat flour).

In some preferred embodiments, the LPC compositions are incorporatedinto chewable matrices. Preferred chewable matrices jelly candies andgelatin-based gummi candy. Exemplary gummi candies include gummi bears,gummi worms, gummi frogs, gummi hamburgers, gummi cherries, gummi sodabottles, gummi sharks, gummi army men, gummi hippopotami, gummilobsters, gummi watermelons, gummi octopuses, gummi apples, gummipeaches, and gummi oranges. The terms “gummi” and “gummy” are usedinterchangeably herein.

In some embodiments, the present invention provides compositionscomprising the LPC compositions described above and one or moreadditional omega-3 fatty acid derivatives or free fatty acids. Theomega-3 fatty acid derivatives or free fatty acids may be derived fromthe neutral lipid extract or from an additional source, such as fish oilor omega-3 ester composition. In some embodiments, the one or moreadditional omega-3 fatty acid derivatives are selected from omega-3esters and glycerides. For example, in some embodiments, the compositionmay comprise from about 1% to about 60% w/w of the krill oil composition(i.e., weight of phospholipid compounds/total weight of composition),with the remaining 99% to 40% w/w of the composition being omega-3glycerides, esters, or free fatty acids or a combination thereof (i.e.,weight of omega-3 glycerides, esters, or free fatty acids or acombination thereof/total weight of the composition). In someembodiments, the composition may comprise from about 5% to about 60% w/wphospholipids, with the remaining 95% to 40% w/w of the compositionbeing omega-3 glycerides, esters, or free fatty acids or a combinationthereof. In some embodiments, the composition may comprise from about20% to about 60% w/w phospholipids, with the remaining 80% to 40% w/w ofthe composition being omega-3 glycerides, esters, or free fatty acids ora combination thereof. In some embodiments, the composition may comprisefrom about 30% to about 60% w/w phospholipids, with the remaining 70% to40% w/w of the composition being omega-3 glycerides, esters, or freefatty acids or a combination thereof. In some embodiments, thecomposition may comprise from about 40% to about 60% w/w phospholipids,with the remaining 60% to 40% w/w of the composition being omega-3glycerides, esters, or free fatty acids or a combination thereof. Insome embodiments, the composition may comprise from about 50% to about60% w/w phospholipids, with the remaining 50% to 40% w/w of thecomposition being omega-3 glycerides, esters, or free fatty acids or acombination thereof.

The LPC compositions of the present invention may further beincorporated into animal and fish feeds and rations. Many different feedrations may be formulated for animals and fish from many different feedingredients. Rations are generally formulated to provide nutrients inaccordance with National Research Council standards. The feedstuffs usedin the ration are chosen according to market price and availability.Thus, some components of the ration may change over time. In the feedsof the present invention, the ration will always contain an LPCcomposition of the invention, preferably in an amount of from 0.1% to50%, 0.5% to 50%, 1.0% to 40%, 1.0% to 30%, 1.0% to 20%, 1.0% to 10%,0.1% to 10%, or 0.5% to 5% of the total fat in the ration. Fordiscussions on feed ration formulation, actual rations and NRCguidelines, see Church, Livestock Feeds and Feeding, O&B Books, Inc.,Corvallis, Oreg. (1984) and Feeds and Nutrition Digest, Ensminger,Oldfield and Heineman eds., Ensminger Publishing Corporation, Clovis,Calif. (1990), incorporated herein by reference.

The animal feed rations of the present invention may be characterizedaccording to NRC requirements. NRC requirements may be found in Church,Livestock Feeds and Feeding, O&B Books, Inc., Corvallis, Oreg. (1984),or other nutritional standards. Animal and fish rations aretraditionally balanced using the protein and energy requirements, andthen adjusted if needed to meet the other requirements. The animal andfish feeds of the present invention will contain about 0.05% to 5%lipids plus other feed materials necessary to balance the feed to meetthe NRC requirements (or other recognized requirements) for thedifferent stages of growth and maintenance.

The relative amounts of protein and energy are adjusted to reflectstandard requirements. The amounts of feed components will vary with thestage of animal fed. A growing ration for young animals and fish willhave higher protein levels, while a finishing ration for finishinganimals for market will have higher energy values which are supplied bycarbohydrates. For example, prestarter, starter and grower-finisherrations for various animals will generally contain about 20-24% protein,18-20% protein and 13-17% protein respectively. In some feedingsituations, care must be taken to provide the appropriate amino acids aswell as overall protein content. Energy requirements may also be met byaddition of fat to the ration. In the present invention, thelysophospholipid composition provides part of the energy requirement.

Typical salmon rations of the invention comprise from about 5% to 65%fish meal and//or krill meal, 5% to 30% vegetable oil and 5%-15% fishoil, expressed as % weight of component/weight of the ration (% w/w) andfrom 0.5% to 5% of an LPC composition of the present invention toprovide a total fat content of from 10% to 45% w/w of the ration. Insome embodiments, the rations have a crude protein content of from about32% to 46%, preferably from about 36% to 42%, a crude lipid content offrom about 26% to 42%, preferably from about 28% to 38%, a carbohydrate(NFE) content of from about 11% to 18%, preferably from about 13% to15%, a fiber content of from about 1% to 5%, preferably from about 1.5%to 2.5%, an ash content of from about 4% to 7%, preferably about 4.5% to6.5%, a total phosphorus content (P) of from about 0.5% to 1.1%,preferably about 0.6% to 1.0%, a gross energy content of from about 20to 30 MJ/kg, preferably from about 23 to 28 MJ/kg, and a digestibleenergy content of from about 20 to 24 MJ/kg.

Other ingredients may be added to the feed ration. These ingredientsinclude, but are not limited to, mineral supplements such as calcium,phosphorus, salt, selenium and zinc; vitamin supplements such asVitamins A, B, D, E, and K; amino acid supplements such as lysine;coccidiostats, except in hog feeds, or growth promoters such asbacitracin or virginamycin; and other active drugs such aschlortetracycline, sulfathiozole, and penicillin. For vitamin, mineraland antibiotic supplement formulation see Church, Livestock Feeds andFeeding, O&B Books, Inc., Corvallis, Oreg. (1984).

In a preferred embodiment, the lysophospholipid compositions areincorporated into a pelleted feed for administration to domesticanimals. Pelleted feed is created by first mixing feed components andthen compacting and extruding the feed components through a die withheat and pressure. The feed is pelleted by methods known in the art,which are described in MacBain, Pelleting Animal Feed, American FeedManufacturers Association, Arlington, Va. (1974), incorporated herein byreference. When incorporating added fat into pelleted feed, caution isneeded in order to avoid making mealy pellets. Generally, only about 2%of the fat is added during pelleting, with the rest added after thepellets have cooled.

The oil and the feed containing the oil may be stabilized by theaddition of antioxidants. Therefore, antioxidants may be added aschemical preservatives in accordance with F.D.A. regulations as listedin the 1997 Official Publication, Association of Feed Control OfficialsIncorporated (1997), herein incorporated by reference. Suitableantioxidants include, but are not limited to: Lecithin, tocopherols,ascorbate, ascorbyl palmitate and spice extracts such as rosemaryextract.

The feeds are formulated as above, and tailored to the requirements ofthe animal to be fed in accordance with NRC guidelines. For example,feeds may be formulated for dogs, cats, poultry, cattle, shrimp, andfish such as salmon, trout, catfish and tilapia. Various feedformulations, balancing methods and requirements for these animals arediscussed in Church, Livestock Feeds and Feeding, O&B Books, Inc.,Corvallis, Oreg. (1984) and Feeds and Nutrition Digest, Ensminger,Oldfield and Heineman eds., Ensminger Publishing Corporation, Clovis,Calif. (1990), incorporated herein by reference.

6. Uses of Krill Phospholipid Compositions

In some embodiments, lysophospholipid compositions of the presentinvention are provided for use in increasing the amount of EPA and/orDHA in a target tissue or organ by oral administration of thelysophospholipid composition. Preferred target tissues and organaccording to the invention are adrenal gland, blood, bone, bone marrow,brain, fat (white), kidney (whole), large intestine mucosa, liver, lung,muscle, myocardium, pancreas, pituitary gland, prostate gland, skin,small intestine mucosa, spleen, stomach mucosa, testis, thymus, and/orthyroid gland.

In some embodiments, an effective amount of the compounds orcompositions described above are administered to a subject in needthereof to treat, prevent, or improve cognition and/or a cognitivedisease, disorder or impairment (memory, concentration, learning(deficit)), or to treat or prevent neurodegenerative disorders. In someembodiments, the cognitive disease, disorder or impairment is selectedfrom Attention Deficit Disorder (ADD), Attention Deficit HyperactivityDisorder (ADHD), autism/autism spectrum disorder (ASD), (dyslexia,age-associated memory impairment and learning disorders, amnesia, mildcognitive impairment, cognitively impaired non-demented, pre-Alzheimer'sdisease, Alzheimer's disease, epilepsy, Pick's disease, Huntington'sdisease, Parkinson disease, Lou Gehrig's disease, pre-dementia syndrome,Lewy body dementia, dentatorubropallidoluysian atrophy, Freidreich'sataxia, multiple system atrophy, types 1, 2, 3, 6, 7 spinocerebellarataxia, amyotrophic lateral sclerosis, familial spastic paraparesis,spinal muscular atrophy, spinal and bulbar muscular atrophy, age-relatedcognitive decline, cognitive deterioration, moderate mental impairment,mental deterioration as a result of ageing, conditions that influencethe intensity of brain waves and/or brain glucose utilization, stress,anxiety, concentration and attention impairment, mood deterioration,general cognitive and mental well-being, neurodevelopmental,neurodegenerative disorders, hormonal disorders, neurological imbalanceor any combinations thereof. In a specific embodiment, the cognitivedisorder is memory impairment.

In some embodiments, an effective amount of the compounds orcompositions described above are administered to a subject in needthereof to treat or prevent a cardiovascular disorder or metabolicsyndrome. In some embodiments, the cardiovascular disorder is selectedfrom atherosclerosis, arteriosclerosis, coronary heart (carotid artery)disease (CHD or CAD), acute coronary syndrome (or ACS), valvular heartdisease, aortic and mitral valve disorders, arrhythmia/atrialfibrillation, cardiomyopathy and heart failure, angina pectoris, acutemyocardial infarction (or AMI), hypertension, orthostatic hypotension,shock, embolism (pulmonary and venous), endocarditis, diseases ofarteries, the aorta and its branches, disorders of the peripheralvascular system (peripheral arterial disease or PAD), Kawasaki disease,congenital heart disease (cardiovascular defects) and stroke(cerebrovascular disease), dyslipidemia, hypertriglyceridemia,hypertension, heart failure, cardiac arrhythmias, low HDL levels, highLDL levels, stable angina, coronary heart disease, acute myocardialinfarction, secondary prevention of myocardial infarction,cardiomyopathy, endocarditis, type 2 diabetes, insulin resistance,impaired glucose tolerance, hypercholesterolemia, stroke,hyperlipidemia, hyperlipoproteinemia, chronic kidney disease,intermittent claudication, hyperphosphatemia, omega-3 deficiency,phospholipid deficiency, carotid atherosclerosis, peripheral arterialdisease, diabetic nephropathy, hypercholesterolemia in HIV infection,acute coronary syndrome (ACS), non-alcoholic fatty liverdisease/non-alcoholic steatohepatitis (NAFLD/NASH), arterial occlusivediseases, cerebral atherosclerosis, arteriosclerosis, cerebrovasculardisorders, myocardial ischemia, coagulopathies leading to thrombusformation in a vessel and diabetic autonomic neuropathy.

In some embodiments, an effective amount of the compounds orcompositions described above are administered to a subject in needthereof to inhibit, prevent, or treat inflammation or an inflammatorydisease. In some embodiments, the inflammation or inflammatory diseaseis selected from organ transplant rejection; reoxygenation injuryresulting from organ transplantation (see Grupp et al., J. Mol. Cell.Cardiol. 31: 297-303 (1999)) including, but not limited to,transplantation of the following organs: heart, lung, liver and kidney;chronic inflammatory diseases of the joints, including arthritis,rheumatoid arthritis, osteoarthritis and bone diseases associated withincreased bone resorption; inflammatory bowel diseases (IBD) such asileitis, ulcerative colitis (UC), Barrett's syndrome, and Crohn'sdisease (CD); inflammatory lung diseases such as asthma, acuterespiratory distress syndrome (ARDS), and chronic obstructive pulmonarydisease (COPD); inflammatory diseases of the eye including cornealdystrophy, trachoma, onchocerciasis, uveitis, sympathetic ophthalmitisand endophthalmitis; chronic inflammatory diseases of the gum, includinggingivitis and periodontitis; inflammatory diseases of the kidneyincluding uremic complications, glomerulonephritis and nephrosis;inflammatory diseases of the skin including sclerodermatitis, psoriasisand eczema; inflammatory diseases of the central nervous system,including chronic demyelinating diseases of the nervous system, multiplesclerosis, AIDS-related neurodegeneration and Alzheimer's disease,infectious meningitis, encephalomyelitis, Parkinson's disease,Huntington's disease, Epilepsy, amyotrophic lateral sclerosis and viralor autoimmune encephalitis, preeclampsia; chronic liver failure, brainand spinal cord trauma, and cancer. The inflammatory disease can also bea systemic inflammation of the body, exemplified by gram-positive orgram negative shock, hemorrhagic or anaphylactic shock, or shock inducedby cancer chemotherapy in response to proinflammatory cytokines, e.g.,shock associated with proinflammatory cytokines. Such shock can beinduced, e.g., by a chemotherapeutic agent that is administered as atreatment for cancer. Other disorders include depression, obesity,allergic diseases, acute cardiovascular events, muscle wasting diseases,and cancer cachexia. Also, inflammation that results from surgery andtrauma can be treated with the phospholipid compositions.

In some embodiments, the LPC compositions described above areadministered to a subject in need thereof to treat a disease orcondition associated with red blood cells and cell membranes, and inparticular a disease or conditions associated with an abnormality in redblood cells of cell membranes. In some embodiments, the condition ordisease is sickle cell disease, sickle cell anemia, or sickle celltrait. In some embodiments, the condition or disease is thalassemia(alpha-, beta- or delta-), thalassemia in combination with ahemoglobinopathy (Hemoglobin E, Hemoglobin S, or Hemoglobin C),splenomegaly, or membrane abnormities such as acanthocytes or spur/spikecells, codocytes (target cells), echinocytes (burr cells), elliptocytesand ovalocytes, spherocytes, stomatocytes (mouth cells) and degmacytes(“bite cells”).

In some embodiments, the effective amount comprises from about 0.1 toabout 5 grams of the krill phospholipid composition, preferably fromabout 0.2 to about 3 grams of the krill phospholipid composition, andmost preferably about 0.5 to about 1.5 grams of the krill phospholipidcomposition.

The krill lysophospholipid compositions of the present invention may beused to treat a variety of subjects. Suitable subjects include humans aswell as domestic animals (such as cattle, horses, sheep, pigs, goats,fish and shrimp), non-human primates, and companion animals (such asdogs, cats and birds). In some preferred embodiments, the subject is ahuman subject of less than 10 years of age, more preferably less than 1year of age, even more preferably less than 1 month of age, and mostpreferably a newborn. In some preferred embodiments, the human subjectis from about 10 to 20 years of age. In some preferred embodiments, thehuman subject is from about 20 to 50 years of age. In some preferredembodiments, the human subject is from about 50 to 100 years of age. Insome preferred embodiments, the human subject is from about 60 to 100years of age. In some preferred embodiments, the human subject is fromabout 70 to 100 years of age.

EXPERIMENTAL Example 1—SUPERBA™ BOOST™

This example describes the production of a LPC composition usingSUPERBA™ BOOST® (Aker Biomarine AS, Lysaker, NO) as the startingphospholipid source. The SUPERBA™ BOOST™ is preferably produced by theSMB processes described elsewhere herein. SUPERBA™ BOOST™ is a krillphospholipid composition wherein 1000 mg contains 560 mg phospholipids,150 mg EPA and 70 mg DHA. Briefly, 9 grams of SUPERBA™ BOOST™ is mixedinto 90 ml EtOH and then 450 ml water is added in one liter round bottomreaction flask purged with nitrogen. Next, 0.40 ml LECITASE™ Ultra isadded and the reaction is allowed to proceed for 30 minutes withstirring. 500 ml EtOH is then added to deactivate the enzyme and themixture is evaporated to dryness using a rotovap. Next, a solvent-basedseparation is performed. The enzyme-treated krill lipid sample is mixedwith a solvent bath with heptane and methanol. The LPC and PC speciesmigrate into the polar solvent (methanol) and the nonpolar lipidspartition into the heptane. Decanting the heptane phase leaves a polarphase with high amount of polar lipids including LPC and PC. 250 ml eachheptane and MeOH are added to the dried lipid composition in a flask.The flask is shaken vigorously, and the phases allowed to separate. Theupper nonpolar heptane phase is decanted and another 250 ml heptane isadded to the polar MeOH phase and the separation repeated. 10 gramssilica gel is then added to the MeOH extract and the solution isevaporated to dryness in a rotovap. The LPC is then purified from thedried lipid composition by flash chromatography (100 g Silica gel in a 5cm diameter column). The LPC is eluted with a series of mobile phases:MPA (heptane); MPB (Toluene:Methanol:Triethylamine, 60:40:1); andMethanol:Triethylamine, 95:5). The fractions are collected andevaporated to dryness.

An LPC composition produced by methods of the present invention wasanalyzed by ¹H-, ³¹P-, 2D-NMR, TLC, GC and HPLC as appropriate. ¹H-NMRand HPLC results are shown in FIG. 1 and FIG. 2, respectively, denotedAKB6444-1.

³¹P- and 2D-NMR were used to determine the phospholipid components theresults are presented in Table 1, Table 2 and Table 3 denoted AKB6444-1.

Example 2—SUPERBA™ BOOST™ Pre-WASH & FLASH

This example describes the production of a LPC composition usingSUPERBA™ BOOST™ (Aker Biomarine AS, Lysaker, NO) as the startingphospholipid source. The SUPERBA™ BOOST™ is preferably produced by theSMB processes described elsewhere herein. SUPERBA™ BOOST™ is a krillphospholipid composition wherein 1000 mg contains 560 mg phospholipids,150 mg EPA and 70 mg DHA. Briefly, 10 grams of SUPERBA™ BOOST™ is mixedinto 100 ml EtOH and then 450 ml water is added in one-liter roundbottom reaction flask purged with nitrogen. Next, 0.40 ml LECITASE™Ultra is added and the reaction is allowed to proceed for 40 minuteswith stirring. 500 ml EtOH is then added to deactivate the enzyme andthe mixture is evaporated to dryness using a rotovap. Next, asolvent-based separation is performed. The enzyme-treated krill lipidsample is mixed with a solvent bath with heptane and methanol. The LPCand PC species migrate into the polar solvent (methanol) and thenonpolar lipids partition into the heptane. Specifically, to the driedsample was added methanol (250 ml) and heptane (250 ml). After vigorousshaking the phases were separated and the methanol phase extracted withanother portion of heptane (250 ml). The heptane fractions were analyzedby TLC and evaporated separately. To the methanol residue was addedsilica gel 60 (20 g) and the solution concentrated to dryness. The LPCis then purified from the dried lipid composition by flashchromatography. The silica gel was loaded onto a column (diameter 5 cm)with silica gel (130 g) and the column eluted. Fraction volume 25 ml.Eluents: 500 ml EtOAc, 250 ml EtOAc:MeOH 80:20, 250 ml EtOAc:MeOH 50:50,250 ml MeOH, 500 ml MeOH:Et3N 95:5, 500 ml MeOH:Et3N 90:10. Fractionswere analyzed by TLC and evaporated according to the findings. LPCcontaining fractions after chromatographic separation yielded a finalmass of 3.28 grams.

An LPC composition produced by methods of the present invention wasanalyzed by ¹H-, ³¹P, 2D-NMR, TLC, GC and HPLC as appropriate. ¹H-NMRand HPLC results are shown in FIG. 7 and FIG. 8, respectively, denotedAKB70005-4.

³¹P- and 2D-NMR were used to determine the phospholipid components theresults are presented in Table 5, Table 9 and Table 10 denotedAKB70005-4.

Example 3—SUPERBA™ BOOST™ FLASH

This example describes the production of a LPC composition usingSUPERBA™ BOOST™ (Aker Biomarine AS, Lysaker, NO) as the startingphospholipid source. The SUPERBA™ BOOST™ is preferably produced by theSMB processes described elsewhere herein. SUPERBA™ BOOST™ is a krillphospholipid composition wherein 1000 mg contains 560 mg phospholipids,150 mg EPA and 70 mg DHA. Briefly, 10 grams of SUPERBA™ BOOST™ is mixedinto 100 ml EtOH and then 450 ml water is added in one liter roundbottom reaction flask purged with nitrogen. Next, 0.40 ml LECITASE™Ultra is added and the reaction is allowed to proceed for 40 minuteswith stirring. 500 ml EtOH is then added to deactivate the enzyme andthe mixture is evaporated to dryness using a rotovap. Next, the LPC isthen purified from the dried lipid composition by flash chromatographyas follows: The residue was re-dissolved in methanol (200 ml) and silicagel 60 (20 g) added and the suspension concentrated to dryness on therotary evaporator. The silica gel was loaded onto a column (diameter 5cm) with silica gel (130 g) and the column eluted. Fraction volume 25ml. Eluents: 500 ml EtOAc, 250 ml EtOAc:MeOH 80:20, 250 ml EtOAc:MeOH50:50, 250 ml MeOH, 500 ml MeOH:Et3N 90:10. Fractions were analyzed byTLC and evaporated according to the findings. LPC containing fractionsafter chromatographic separation yielded a final mass of 3.73 grams.

An LPC composition produced by methods of the present invention wasanalyzed by ¹H-, ³¹P-, 2D-NMR, TLC, GC and HPLC as appropriate. ¹H-NMRand HPLC results are shown in FIG. 9 and FIG. 10, respectively, denotedAKB70005-5.

³¹P- and 2D-NMR were used to determine the phospholipid components theresults are presented in Table 6, Table 9 and Table 10 denotedAKB70005-5.

Example 4—ROMEGA™ Pre-Wash & FLASH

This example describes the production of a LPC composition using ROMEGA™(Arctic Nutrition AS, Ørsta, NO) as the starting phospholipid source.The ROMEGA™ is preferably produced by processes described elsewhere.ROMEGA™ is a herring roe oil and fish oil composition wherein 1000mg/3000 mg contains 340 mg/1020 mg phospholipids, 100 mg/300 mg EPA and320 mg/960 mg DHA. Briefly, 9 grams of ROMEGA™ is mixed into 90 ml EtOHand then 450 ml water is added in one liter round bottom reaction flaskpurged with nitrogen. Next, 0.40 ml LECITASE™ Ultra is added and thereaction is allowed to proceed for 40 minutes with stirring. 500 ml EtOHis then added to deactivate the enzyme and the mixture is evaporated todryness using a rotovap. Next, a solvent-based separation is performed.The enzyme-treated herring roe/fish lipid sample is mixed with a solventbath with heptane and methanol. The LPC and PC species migrate into thepolar solvent (methanol) and the nonpolar lipids partition into theheptane. Specifically, to the dried sample was added methanol (250 ml)and heptane (250 ml). After vigorous shaking the phases were separatedand the methanol phase extracted with another portion of heptane (250ml). The heptane fractions were analyzed by TLC and evaporatedseparately. To the methanol residue was added silica gel 60 (20 g) andthe solution concentrated to dryness. The LPC is then purified from thedried lipid composition by flash chromatography. The silica gel wasloaded onto a column (diameter 5 cm) with silica gel (130 g) and thecolumn eluted. Fraction volume 25 ml. Eluents: 500 ml EtOAc, 250 mlEtOAc:MeOH 80:20, 250 ml EtOAc:MeOH 50:50, 250 ml MeOH, 500 ml MeOH:Et3N95:5, 500 ml MeOH:Et3N 90:10. Fractions were analyzed by TLC andevaporated according to the findings. LPC containing fractions afterchromatographic separation yielded a final mass of 0.84 grams.

An LPC composition produced by methods of the present invention wasanalyzed by ¹H-, ³¹P, 2D-NMR, TLC, GC and HPLC as appropriate. ¹H-NMRand HPLC results are shown in FIG. 5 and FIG. 6, respectively, denotedAKB70005-3.

³¹P- and 2D-NMR were used to determine the phospholipid components theresults are presented in Table 5, Table 7 and Table 8 denotedAKB70005-3.

Example 5—ROMEGA™ FLASH

This example describes the production of a LPC composition using ROMEGA™(Arctic Nutrition AS, Ørsta, NO) as the starting phospholipid source.The ROMEGA™ is preferably produced by a process described elsewhere.ROMEGA™ is a herring roe oil and fish oil composition wherein 1000mg/3000 mg contains 340 mg/1020 mg phospholipids, 100 mg/300 mg EPA and320 mg/960 mg DHA. Briefly, 9 grams of ROMEGA™ is mixed into 90 ml EtOHand then 450 ml water is added in one-liter round bottom reaction flaskpurged with nitrogen. Next, 0.40 ml LECITASE™ Ultra is added and thereaction is allowed to proceed for 40 minutes with stirring. 500 ml EtOHis then added to deactivate the enzyme and the mixture is evaporated todryness using a rotovap. Next, the LPC is then purified from the driedlipid composition by flash chromatography as follows: The residue wasre-dissolved in methanol (200 ml) and silica gel 60 (20 g) added and thesuspension concentrated to dryness on the rotary evaporator. The silicagel was loaded onto a column (diameter 5 cm) with silica gel (130 g) andthe column eluted. Fraction volume 25 ml. Eluents: 500 ml EtOAc, 250 mlEtOAc:MeOH 80:20, 250 ml EtOAc:MeOH 50:50, 250 ml MeOH, 500 ml MeOH:Et3N90:10. Fractions were analyzed by TLC and evaporated according to thefindings. LPC containing fractions after chromatographic separationyielded a final mass of 0.76 grams.

An LPC composition produced by methods of the present invention wasanalyzed by 1H-, 31P-, 2D-NMR, TLC, GC and HPLC as appropriate. ¹H-NMRand HPLC results are shown in FIG. 5 and FIG. 6, respectively, denotedAKB70005-2.

³¹P- and 2D-NMR were used to determine the phospholipid components theresults are presented in Table 4, Table 7 and Table 8 denotedAKB70005-2.

TABLE 1 Results (summary), 2D- and 31P-NMR (PLs) of sample AKB69444-1Component Method Weight Percent PC ³¹P-NMR 3.04 — 1-LPC ³¹P-NMR 5.73 —2-LPC ³¹P-NMR 75.38 — PI ³¹P-NMR 0.00 — PS-Na ³¹P-NMR 0.00 — PE ³¹P-NMR0.00 — LPE ³¹P-NMR 0.00 — APE ³¹P-NMR 0.00 — PG ³¹P-NMR 0.00 — DPG³¹P-NMR 0.00 — PA ³¹P-NMR 0.00 — LPA ³¹P-NMR 0.00 — other PL ³¹P-NMR0.34 — sum ³¹P-NMR 84.15 — phosphorus ³¹P-NMR 4.83 —

TABLE 2 Results (summary), 2D- and 31P-NMR (ether PLs) of sampleAKB69444-1 Component Method Weight Percent PC ³¹P-NMR 1.90 — PC-ether³¹P-NMR 1.10 — 2-LPC ³¹P-NMR 57.20 — 2-LPC-ether ³¹P-NMR 18.20 — otherPL ³¹P-NMR 0.34 — sum ³¹P-NMR 84.15 — phosphorus ³¹P-NMR 4.83 —

TABLE 3 The LPC composition contained 38.74% omega-3 fatty acids w/w ofthe composition as determined by ¹H-NMR of sample AKB69444-1. InitialInitial Test Integral weight Integral weight mMoi MMoi Content ContentItem TI [mg] TI IS [mg] IS IS TI [mg] TI [%] TI AKB67919-1 198.61 350.6590.00 20.43 0.0626 0.4141 135.8263 38.74 Int. Standard Molecular weightTI 328.00 TPP Molecular weight IS 326.29 Rounding Number of atoms TI 3 4Number of atoms IS 9 Balance XPE-841 en Content [%] IS 99.9Mettlet-Toledo XPE205DR/M Comment: Initial weight is higher thanrequired MinWeigh of 10 mg. *) calculated as DHA. contains mixture ofany w-3 FA (omega 3 fatty acids)

TABLE 4 Omega-3 fatty acids w/w of the LPC composition as determined by¹H-NMR for the samples AKB70005-1 and AKB70005-2. AKB70005-1specification AKB70005-2 specification Romega Lecitase Romega testAB:ABM-1:1 AB:ABM-1:3 Flash components method weight-% weight-% omega 3FA *) ¹H-NMR 43.56 — 43.78 — *) calculated as DHA, contains mixture ofany w-3 FA (omega 3 fatty acids)

TABLE 5 Omega-3 fatty acids w/w of the LPC composition as determined by¹H-NMR for the samples AKB70005-3 and AKB70005-4. AKB70005-3specification AKB70005-4 specification Lecitase Romega Lecitase Boosttest MS:ABM-1:47 pre/flash MS:ABM-1:43B pre/flash components methodweight-% weight-% omega 3 FA *) ¹H-NMR 42.85 — 47.55 — *) calculated asDHA, contains mixture of any w-3 FA (omega 3 fatty acids)

TABLE 6 Omega-3 fatty acids w/w of the LPC composition as determined by¹H-NMR for the sample AKB70005-5. AKB70005-5 specification LecitaseBoost test MS:ABM-1:45B flash components method weight-% omega 3 FA *)¹H-NMR 47.06 — *) calculated as DHA, contains mixture of any w-3 FA(omega 3 fatty acids)

TABLE 7 Results (summary), 2D- and 31P-NMR (PLs) for AKB70005-1,AKB70005-2 and AKB70005-3. AKB70005-1 specification AKB70005-2specification AKB70005-3 specification Romega Lecitase Romega LecitaseRomega test AB:ABM-1:1 AB:ABM-1:3 Flash MS:ABM-1:47 pre/flash componentsmethod weight % weight % weight % PC ³¹P-NMR 20.97 — 5.03 — 7.29 — 1-LPC³¹P-NMR 0.25 — 6.66 — 6.41 — 2-LPC ³¹P-NMR 2.06 — 59.95 — 56.42 — PI³¹P-NMR 0.35 — 0.00 — 0.00 — SPH ³¹P-NMR 0.69 — 3.70 — 4.34 — PE ³¹P-NMR1.73 — 0.00 — 0.00 — LPE ³¹P-NMR 0.25 — 0.00 — 0.00 — APE ³¹P-NMR 0.00 —0.00 — 0.00 — PG ³¹P-NMR 0.00 — 0.00 — 0.00 — DPG ³¹P-NMR 0.00 — 0.00 —0.00 — PA ³¹P-NMR 0.09 — 0.00 — 0.26 — LPA ³¹P-NMR 0.00 — 0.00 — 0.00 —other PL ³¹P-NMR 0.00 — 0.55 — 0.00 — sum ³¹P-NMR 26.39 — 75.88 — 74.73— phosphorus ³¹P-NMR 1.06 — 4.21 — 4.10 —

TABLE 8 Results (summary), 2D- and 31P-NMR (PLs) for AKB70005-1,AKB70005-2 and AKB70005-3. AKB70005-1 specification AKB70005-2specification AKB70005-3 specification Romega Lecitase Romega LecitaseRomega test AB:ABM-1:1 AB:ABM-1:3 Flash MS:ABM-1:47 pre/flash componentsmethod weight % weight % weight % PC ³¹P-NMR 20.20 — 2.40 — 3.50 —PC-ether ³¹P-NMR 0.80 — 2.70 — 3.80 — 2-LPC ³¹P-NMR 1.40 — 58.50 — 55.10— 2-LPC-ether ³¹P-NMR 0.60 — 1.00 — 0.80 — 2-LPC-plasma ³¹P-NMR 0.00 —0.50 — 0.50 — PE ³¹P-NMR 1.60 — 0.00 — 0.00 — PE-ether ³¹P-NMR 0.20 —0.00 — 0.00 —

TABLE 9 Results (summary), 2D- and 31P-NMR (PLs) for AKB70005-4 andAKB70005-5. AKB70005-4 specification AKB70005-5 specification LecitaseBoost Lecitase Boost test MS:ABM-1:43B pre/flash MS:ABM-1:45B flashcomponents method weight-% weight-% PC ³¹P-NMR 9.24 — 3.44 — 1-LPC³¹P-NMR 7.24 — 7.37 — 2-LPC ³¹P-NMR 63.04 — 68.64 — PI ³¹P-NMR 0.00 —0.00 — PS-Na ³¹P-NMR 0.00 — 0.00 — PE ³¹P-NMR 0.00 — 0.00 — LPE ³¹P-NMR0.00 — 0.00 — APE ³¹P-NMR 0.00 — 0.00 — PG ³¹P-NMR 0.00 — 0.00 — DPG³¹P-NMR 0.00 — 0.00 — PA ³¹P-NMR 0.00 — 0.00 — LPA ³¹P-NMR 0.00 — 0.00 —other PL ³¹P-NMR 0.00 — 0.00 — sum ³¹P-NMR 79.52 — 79.45 — phosphorus³¹P-NMR 4.43 — 4.54 —

TABLE 10 Results (summary), 2D- and 31P-NMR (PLs) for AKB70005-4 andAKB70005-5. AKB70005-4 specification AKB70005-5 specification LecitaseBoost Lecitase Boost MS:ABM-1:43B pre/flash MS:ABM-1:45B flashcomponents test method weight-% weight-% PC ³¹P-NMR 4.70 — 1.90 —PC-ether ³¹P-NMR 4.60 — 1.60 — 2-LPC ³¹P-NMR 60.30 — 65.50 — 2-LPC-ether³¹P-NMR 1.70 — 1.60 — 2-LPC-plasma ³¹P-NMR 1.00 — 1.60 — PE ³¹P-NMR 0.00— 0.00 — PE-ether ³¹P-NMR 0.00 — 0.00 —

TABLE 11 Legend (abbreviations). abbreviation full name 1-LPC1-lyso-phosphatidylcholine 2-LPC 2-lyso-phosphatidylcholine APEN-acyl-phosphatidylethanolamine CDCl₃ chloroform-d₁ Cs₂CO₃ caesiumcarbonate DHA docosahexaenoic acid D₂O deuteriumoxide DPGdiphosphatidylglycerol EDTA ethylenediaminetetraacetic FA fatty acid ISinternal standard LPA lyso-phosphatidic acid LPIlyso-Phosphatidylinositol LPS lyso-Phosphatidylserine MeOD methanol-d₄NMR nuclear magnetic resonance PA phosphatidic acid PCphosphatidylcholine PE phosphatidylethanolamine PG phosphatidylglycerolPI phosphatidylinositol PL phospholipid PS phosphatidylserine SPHsphingomyelin TMS tetramethylsilane TPP triphenyl phosphate w-3 FA omega3 fatty acid

Example 6

An LPC composition was produced using SUPERBA™ krill oil as a startingpoint. The phospholipid content was analyzed by ³¹P-NMR. The results arepresented in Table 12.

TABLE 12 Phospholipid Weight-% Mol-% MW [g/mol] PC 7.73 15.08 790.01-LPC 10.23 29.50 534.5 2-LPC 17.51 50.47 534.5 PI —*) —*) 907.0 LPI —*)—*) 629.5 PS-Na —*) —*) 833.0 LPS —*) —*) 555.5 SPH —*) —*) 812.0 PE0.48 0.96 770.0 LPE 0.96 3.00 492.5 APE —*) —*) 1032.0 PG —*) —*) 820.0DPG —*) —*) 774.0 PA —*) —*) 746.0 LPA —*) —*) 468.5 Other PL 0.52 0.99812.0 Sum 37.44 100.00 Phosphorus 2.01 Comment: Integrals ofPhospholipid signals, which are not evaluable or belong to not listedPhospholipids, are recorded as “other”. *)-not observed, no signalassignment

Example 7

This example provides a summary of production of furtherlysophospholipid compositions of the invention.

Method of Synthesizing LPC Species from Krill OilThe methods used to synthesize LPC from krill oil may be divided in foursteps depending on target composition of final mixture/sample:

-   -   1. CRUDE: a process that allows a concentration of 14-27.3        weight % LPC in the final composition to be achieved (Table        13.1-1 and 14.1-1).    -   2. POLAR-1/POLAR-2: a process after CRUDE that allows a        concentration of 35.2-65.7 weight % LPC in the final composition        to be achieved (Table 13.1-2 and 14.1-2).    -   3. FLASH: a process after CRUDE that allows a concentration of        56.6-81.1 weight % LPC in the final composition to be achieved        (Table 13.1-3).    -   4. FORMULATION: a process after POLAR-2 that allows a        concentration of 39.3-41.1 weight % LPC with 5-14 weight %        PEG400 in the final composition to be achieved (14.1-3).        FLASH and POLAR are alternative processes to achieve an LPC        enrichment of CRUDE-1 and CRUDE-2 LPC mixtures/samples, whereas        FORMULATION is a process that allows for a formulation with        POLAR-2 LPC mixtures/samples with PEG. Flow diagrams of the        various processes are provided as FIGS. 11 (Process 1) and 12        (Process 2).

1. Crude Samples:

CRUDE-1 & CRUDE-2 LPC mixtures/samples follow the same processes.Superba Boost (10 g) was dissolved in EtOH (2-10 g), diluted with pHstabilized water (2-45 g, pH 6-12) to achieve a mixture pH of 5.2-6.2,added Lecitase ultra 40 μl, capped and stirred at room temperature for120-1440 minutes. To prevent oxidation, samples may be flushed with N2.The reaction mixture was quenched with addition of EtOH (25-50 g) andconcentrated under reduced pressure at 50° C. to afford the CRUDE-1 andCRUDE-2 LPC-mixtures/samples (Tables 13 and 16, respectively).

2. Polar-1/Polar-2 Samples:

POLAR-2: The POLAR process was initiated directly after quench in theCRUDE process. Thus, the CRUDE sample was not concentrated under reducedpressure at 50° C. to afford the CRUDE-1 and CRUDE-2LPC-mixtures/batches, rather, 25-100 g of heptane was added to theCRUDE-1 or CRUDE-2 mixture to achieve a phase separation and thus anenrichment of polar lipids in the EtOH and water rich phase, called thepolar phase. The heptane phase was decanted leaving only the polarphase. The polar phase was concentrated under reduced pressure to affordthe POLAR-1 and POLAR-2 LPC-mixtures/samples (Tables 14 and 17,respectively).

3. FLASH Samples:

The CRUDE samples were re-dissolved in ethanol (10-100 g) with additionof 25-50 g Silica gel 60 and evaporated to dryness. Flash chromatography(125 g Silicagel 60, column diameter 5 cm) was performed. Elution with500 ml 80:20 MPA:MPB, 500 ml 50:50 MPA:MPB, 500 ml MP B, 500 ml MP C wasperformed. The column was subsequently extruded with EtOH:Et3N (80:20,300 mL). Based on the results different fractions were combined andevaporated to afford the FLASH LPC-mixtures/samples (Table 15).

4. FORMULATION Samples:

A formulation with PEG400 was made of the POLAR mixtures/samples. Forthis, EtOH and PEG400 was added to the POLAR mixture whereby the mixture(EtOH, PEG400 and POLAR LPC mixture) was concentrated under reducedpressure at 50° C. to afford the FORMULATION LPC-mixtures/samples (Table18) with a final concentration of 5-14 weight % PEG400 and 4-7 weight %EtOH (Table 18).

TABLE 13 Analytical results from six CRUDE-1 krill LPC compositionbatches from Process 1 CRUDE-1 Batch number AKB:ABM- AKB:ABM- AKB:ABM-AKB:ABM- MS:ABM- MS:ABM- Parameter 1:7B 1:9B 1:13 1:15 1:65 1:69Phospholipid comp, weight %, ³¹P NMR PC 4.9 5.7 7.8 5.1 4.3 10.8PC-ether 2.8 4.2 4.3 4.2 3.5 4.8 1-LPC 5.0 8.6 6.6 6.3 10.2 10.7 2-LPC9.1 16.6 17.3 20.7 17.2 16.2 2-LPC-ether <0.1 <0.1 <0.1 <0.1 0.3 0.4 PE0.2 0.4 0.2 0.2 <0.1 0.4 PE-ether <0.1 <0.1 <0.1 0.5 0.5 0.4 LPE 0.3 0.70.3 <0.1 1.0 0.9 Other 0.7 0.9 1.0 1.1 0.5 0.4 Sum total PL 23.0 37.137.4 38.1 37.5 45.0 Sum total LPC 14.1 25.2 23.9 27.0 27.7 27.3Phosphorus 1.2 1.9 1.9 2.0 2.1 2.3 Fatty acids, g/100 g, GC 12:0  Not<0.1 <0.1 14:0  Available 3.7 3.9 15:0  0.2 0.2 16:0  12.5 13.3 16:1n72.2 2.3 18:0  0.7 0.7 18:1n9 4.3 4.5 18:1n7 3.6 3.8 18:2n6 0.9 0.918:3n3 1.4 1.5 18:4n3 2.6 2.7 20:1n9 0.3 0.3 20:4n6 0.2 0.2 20:3n3 <0.1<0.1 20:4n3 0.3 0.4 20:5n3 15.2 16.0 22:1n9 0.5 0.5 21:5n3 0.5 0.522:5n3 0.3 0.3 22:6n3 7.6 7.9 Unknown 3.2 3.4 Saturated fatty acids 17.118.1 Monoenic fatty acids 10.9 11.4 PUFA (n-6) fatty 1.1 1.1 acids PUFA(n-3) fatty 27.9 29.2 acids Total-PUFA fatty 29.0 30.3 acids Fatty acidstotal 60.2 63.2

TABLE 14 Analytical results from six POLAR-1 krill LPC compositionbatches from Process 1 POLAR-1 Batch number AKB:ABM- AKB:ABM- AKB:ABM-AKB:ABM- MS:ABM- MS:ABM- Parameter 1:7B 1:9B 1:13 1:15 1:65 1:69Phospholipid comp, weight %, ³¹P NMR PC 11.7 7.8 11.1 7.1 8.5 Notavailable PC-ether 5.0 5.7 6.0 6.0 9.4 1-LPC 8.9 12.4 7.0 9.0 28.8 2-LPC26.3 28.5 33.7 33.8 35.9 2-LPC-ether <0.1 <0.1 <0.1 <0.1 1.0 PE <0.1 0.40.4 0.2 <0.1 PE-ether <0.1 <0.1 <0.1 <0.1 0.9 LPE <0.1 1.1 1.1 0.8 1.8Other 2.1 1.9 2.2 2.2 <0.1 Sum total PL 54.0 57.8 61.4 59.1 86.3 Sumtotal LPC 35.2 40.9 40.7 42.8 65.7 Phosphorus 1.2 1.9 1.9 2.0 4.7 Fattyacids, g/100 g, Not GC 12:0  <0.1 <0.1 <0.1 <0.1 available 14:0  1.1 2.01.4 1.4 15:0  0.1 0.1 0.1 0.1 16:0  6.2 8.5 8.4 7.9 16:1n7 0.9 1.4 1.11.1 18:0  0.6 0.6 0.7 0.7 18:1n9 2.4 3.2 2.7 2.7 18:1n7 1.6 2.3 2.3 2.118:2n6 0.6 0.8 0.7 0.7 18:3n3 1.1 1.5 1.4 1.3 18:4n3 1.3 2.2 2.0 2.020:1n9 0.1 0.2 0.2 0.1 20:4n6 0.2 0.3 0.3 0.2 20:3n3 <0.1 <0.1 <0.1 <0.120:4n3 0.2 0.4 0.4 0.3 20:5n3 11.4 18.8 18.2 17.4 22:1n9 0.2 0.3 0.3 0.321:5n3 0.4 0.6 0.6 0.6 22:5n3 0.2 0.4 0.4 0.3 22:6n3 5.0 9.1 8.8 8.4Unknown 0.7 1.2 1.0 1.0 Saturated fatty acids 7.9 11.2 10.6 10.1Monoenic fatty acids 5.2 7.3 6.5 6.4 PUFA (n-6) fatty 0.8 1.1 1.0 1.0acids PUFA (n-3) fatty 19.6 33.0 31.7 30.3 acids Total-PUFA fatty 20.434.1 32.6 31.3 acids Fatty acids total 34.3 53.9 50.8 48.7

TABLE 15 Analytical results from six FLASH krill LPC composition batchesfrom Process 1 FLASH Batch number MS- MS: MS: MS: MS: MS: MS: ABM1- ABM-ABM- ABM- ABM- ABM- ABM- Parameter 31 1:43B 1:45B 1:65 1:67 1:69 1:71Phospholipid comp, weight %, ³¹P NMR PC 1.9 4.7 1.9 3.3 1.8 13.7 6.7PC-ether 1.1 4.6 1.6 3.8 1.6 5.6 3.1 1-LPC 5.7 7.2 7.4 8.7 8.0 7.0 7.12-LPC 57.2 60.3 65.5 68.5 55.0 48.6 58.3 2-LPC-ether 18.2 2.7 3.2 1.41.2 1.0 1.6 PE <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 PE-ether <0.1 <0.1<0.1 <0.1 <0.1 <0.1 <0.1 LPE <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 Other0.3 <0.1 <0.1 <0.1 0.5 0.6 <0.1 Sum total PL 84.2 79.5 79.5 85.7 68.176.5 76.8 Sum total LPC 81.1 70.2 76.1 78.6 64.2 56.6 67.0 Phosphorus4.8 4.4 4.5 4.9 3.9 4.1 4.3 Fatty acids, Not Not g/100 g, GC availableavailable 12:0  <0.1 <0.1 <0.1 <0.1 <0.1 14:0  0.2 0.2 0.2 0.4 0.3 15:0 <0.1 <0.1 <0.1 <0.1 <0.1 16:0  1.0 1.4 1.4 4.4 3.4 16:1n7 0.6 0.6 0.60.7 0.6 18:0  0.2 0.4 0.5 0.5 0.5 18:1n9 2.5 2.4 2.6 2.7 2.6 18:1n7 0.40.5 0.5 1.2 0.9 18:2n6 0.9 0.9 1.0 0.9 1.0 18:3n3 1.8 1.9 2.0 1.8 1.918:4n3 2.6 2.5 2.6 2.3 2.3 20:1n9 <0.1 <0.1 <0.1 0.1 <0.1 20:4n6 0.3 0.30.3 0.3 0.3 20:3n3 <0.1 <0.1 <0.1 <0.1 <0.1 20:4n3 0.4 0.4 0.4 0.4 0.420:5n3 24.5 24.3 24.3 22.0 21.7 22:1n9 <0.1 <0.1 <0.1 0.2 0.2 21:5n3 0.90.9 0.9 0.8 0.8 22:5n3 0.5 0.5 0.5 0.5 0.5 22:6n3 12.4 10.5 10.3 9.8 9.6Unknown 0.4 0.4 0.6 0.4 0.3 Saturated fatty 1.4 1.9 1.9 5.3 4.2 acidsMonoenic fatty 3.5 3.5 3.7 4.8 4.4 acids PUFA (n-6) 1.2 1.2 1.3 1.2 1.2fatty acids PUFA (n-3) 43.1 40.9 40.9 37.5 37.1 fatty acids Total-PUFA44.3 42.1 42.1 38.6 38.3 fatty acids Fatty acids 49.6 47.9 48.3 49.147.1 total

TABLE 16 Analytical results from two CRUDE-2 krill LPC compositionbatches from Process 2 CRUDE-2 Batch number Parameter LS:ABM_9C0LS:ABM_10C0 Phospholipid comp, weight %, ³¹P NMR PC 4.3 6.1 PC-ether 4.33.6 1-LPC 10.7 4.0 2-LPC 15.3 21.0 2-LPC-ether — — PE 0.7 0.9 PE-ether —— LPE 0.9 1.8 Other 0.9 2.2 Sum total PL 37.1 39.5 Sum total LPC 26.025.0 Phosphorus 2.0 2.1 Total PL gravimetric 42.0 45.3 Total NLgravimetric 58.0 54.7 H₂O %, w/w 1.0 0.5 Fatty acids, g/100 g, GC 12:00.2 0.1 14:0 4.4 4.6 15:0 0.2 0.2 16:0 13.5 14.3 16:1n7 2.1 4.1 18:0 1.31.7 18:1n9 4.8 5.3 18:1n7 4.0 3.8 18:2n6 1.0 0.8 18:3n3 1.7 0.5 18:4n33.7 1.5 20:1n9 0.3 0.4 20:4n6 0.2 0.2 20:3n3 0.1 0.1 20:4n3 0.5 0.320:5n3 16.0 17.8 22:1n9 0.4 0.8 21:5n3 0.5 0.6 22:5n3 0.4 0.3 22:6n3 8.66.8 Unknown 0 0 Saturated fatty acids 19.5 20.9 Monoenic fatty acids11.5 14.4 PUFA (n-6) fatty acids 1.2 1.0 PUFA (n-3) fatty acids 31.427.9 Total-PUFA fatty acids 32.6 28.8 Fatty acids total 63.7 64.1

TABLE 17 Analytical results from eight POLAR-2 krill LPC compositionbatches from Process 2 POLAR-2 Batch number LS:ABM_3 LS:ABM_3 LS:ABM_8LS:ABM_8 LS:ABM_8 LS:ABM_8 LS:ABM_9 LS:ABM_9 Parameter K D A1 B1 Cl D1-1A1 C1 Phospholipid comp, weight %, ³¹P NMR PC 8.4 5.8 7.5 10.8 6.3 12.29.4 8.4 PC-ether 5.5 3.4 7.5 7.9 5.4 5.8 7.9 8.3 1-LPC 15.6 9.4 25.018.0 12.8 11.2 18.6 14.1 2-LPC 33.4 33.0 21.2 23.7 36.6 30.6 29.5 35.62-LPC-ether <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 — PE 1.2 0.9 1.2 1.5 1.11.7 1.2 1.1 PE-ether <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 — LPE 2.8 2.41.6 2.9 2.8 3.0 1.8 1.7 Other 4.6 3.4 1.6 2.0 4.9 4.5 1.8 2.3 Sum totalPL 71.5 58.1 65.7 66.7 70.0 68.9 70.1 71.4 Sum total LPC 49 42.4 46.241.7 49.4 41.8 48.1 49.7 Phosphorus 3.8 3.12 3.5 3.5 3.7 3.6 3.7 3.8Total PL gravimetric 77.2 63.4 76.2 73.7 76.8 79.4 77.0 79.0 Total NLgravimetric 22.8 36.6 23.8 26.3 23.2 20.6 23.0 21.0 H₂O %, w/w Notavailable 2.4 1.4 1.2 3.7 0.9 0.6 Fatty acids, g/100 g, GC Pending 12:0 <0.1 <0.1 <0.1 <0.1 0.1 0.1 14:0  1.4 1.9 1.3 1.4 1.3 1.1 15:0  0.1 0.1<0.1 <0.1 0.1 0.1 16:0  5.8 7.9 6.2 7.3 6.1 5.2 16:1n7 0.9 3.1 2.3 1.80.9 0.8 18:0  0.8 1.0 0.9 1.1 1.0 0.9 18:1n9 2.7 3.3 2.8 3.0 2.6 2.318:1n7 1.7 1.8 1.4 1.9 1.7 1.4 18:2n6 0.8 1.1 0.9 0.7 0.8 0.7 18:3n3 1.50.4 0.5 0.5 1.5 1.4 18:4n3 2.6 1.2 2.0 1.4 2.7 2.5 20:1n9 0.3 0.3 0.30.3 0.1 0.1 20:4n6 0.2 0.4 0.3 0.2 0.2 0.2 20:3n3 0.1 <0.1 <0.1 <0.1 0.10.1 20:4n3 0.4 0.4 0.4 0.3 0.4 0.4 20:5n3 18.8 20.4 21.4 21.5 18.7 16.822:1n9 0.2 0.3 0.4 0.4 0.2 0.2 21:5n3 0.7 0.7 0.8 0.9 0.6 0.6 22:5n3 0.40.5 0.4 0.4 0.4 0.4 22:6n3 10.3 9.2 9.9 8.7 10.1 8.9 Unknown 1.6 2.1 1.71.7 <0.1 <0.1 Saturated fatty acids 8.1 10.8 8.4 9.8 8.6 7.3 Monoenicfatty acids 5.9 8.8 7.1 7.3 5.4 4.8 PUFA (n-6) fatty acids 1.0 1.5 1.20.9 1.0 0.9 PUFA (n-3) fatty acids 34.8 32.7 35.3 33.6 34.5 30.9Total-PUFA fatty acids 35.8 34.1 36.5 34.6 35.5 31.8 Fatty acids total51.4 55.8 53.6 53.3 49.6 44.0

TABLE 18 Analytical results from two Formulation krill LPC compositionbatches from Process 2 FORMULATION Batch number Parameter LS:ABM_9A2LS:ABM_9C2 Phospholipid comp, weight %, ³¹P NMR PC 7.7 6.9 PC-ether 6.46.6 1-LPC 15.0 12.6 2-LPC 24.3 28.5 2-LPC-ether — — PE 1.1 1.1 PE-ether— — LPE 1.3 1.4 Other 1.1 1.4 Sum total PL 57.0 58.5 Sum total LPC 39.341.1 Phosphorus 3.0 3.1 Fatty acids, g/100 g, GC 12:0 0.1 0.1 14:0 1.21.0 15:0 0.1 0.1 16:0 5.3 4.8 16:1n7 0.7 0.7 18:0 1.0 1.0 18:1n9 2.2 2.118:1n7 1.4 1.3 18:2n6 0.7 0.7 18:3n3 1.3 1.3 18:4n3 2.3 2.4 20:1n9 0.10.1 20:4n6 0.2 0.2 20:3n3 0.1 0.1 20:4n3 0.3 0.4 20:5n3 15.8 16.6 22:1n90.2 0.2 21:5n3 0.5 0.6 22:5n3 0.3 0.3 22:6n3 8.4 9.1 Unknown <0.1 <0.1Saturated fatty acids 7.5 6.9 Monoenic fatty acids 4.6 4.4 PUFA (n-6)fatty acids 0.8 0.9 PUFA (n-3) fatty acids 29.2 30.7 Total-PUFA fattyacids 30.1 31.6 Fatty acids total 42.2 42.9

Table 19 provides additional analytical data for the various batches.

TABLE 19 CRUDE-2 batches POLAR-2 batches FORMULATION batches LS:ABM_LS:ABM_ LS:ABM_ LS:ABM_ LS:ABM_ LS:ABM_ 9C0 10C0 9A1 9C1 9A2 9C2 Salt(NaCl) (ppm) 490.4 3262.4 1051.7 941.6 999.5 839.6 Astaxanthin (μg/g)123.2 190.8 75.5 82.8 90.1 74.2 Conductivity 15.6 64.4 20.3 16.3 62.052.8 (μS/cm)

Example 9

This example provides data on the uptake of a lysophospholipidcompositions of the present invention in biological tissues. Briefly,the LS:ABM-9C0 lysophospholipid composition described above was spikedwith ¹⁴C-labelled lysoPC-EPA or lysoPC-DHA and compared to a purifiedkrill PC composition (98% PC formulated with PEG) spiked with¹⁴C-labelled PC-EPA or DHA. The spiked compositions were orallyadministered to rats and the uptake into various tissues was measured byQuantitative Whole Body Autoradiography. Data was collected both on thetiming of uptake in various organs and tissues as well as the amount ofincorporation into the tissue. The results are provided in Tables 20-23.Surprisingly, these data indicate that uptake of both the lysoPC-EPAspiked lysophospholipid composition and lysoPC-DHA spikedlysophospholipid composition was both faster in time and greater intotal amount of EPA and DHA incorporated as compared to the samples thatwere prepared with intact phospholipids (i.e., samples that did notcontain any appreciable amount of lysophospholipids). This result wasobserved in all investigated organs/tissues except the eye.

TABLE 20 Time after administration (hrs) EPA-PC 0.5 3 8 24 72 96 168 336(% of dose/organ) Adrenal gland <0.001 0.009 0.016 0.008 0.011 0.0080.007 0.003 Blood 0.082 0.971 1.012 0.267 0.302 0.269 0.191 0.122 Bone<0.014 0.236 0.414 0.162 0.259 0.234 0.257 0.16 Bone marrow 0.001 0.050.099 0.059 0.108 0.058 0.044 0.017 Brain <0.001 0.01 0.025 0.028 0.0880.102 0.118 0.147 Eye (whole) <0.001 0.002 0.003 0.001 0.006 0.006 0.0040.005 Fat (white) <0.015 0.956 6.599 1.936 2.515 2.811 2.651 0.995Kidney (whole) 0.006 0.127 0.216 0.119 0.195 0.194 0.122 0.069 Largeintestine mucosa <0.002 0.886 0.23 0.162 0.16 0.162 0.108 0.059 Liver0.103 5.526 10.551 3.188 2.472 2.082 1.286 0.0639 Lung 0.005 0.115 0.1810.074 0.106 0.089 0.074 0.038 Muscle <0.094 2.304 4.432 3.47 6.818 7.4178.361 7.175 Myocardium 0.005 0.074 0.097 0.056 0.119 0.145 0.141 0.103Pancreas 0.003 0.126 0.228 0.107 0.156 0.122 0.097 0.058 Pituitary glandn.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. Prostate gland n.d. n.d. n.d.n.d. n.d. n.d. n.d. n.d. Skin 0.037 0.798 2.286 1.172 2.181 2.426 1.2921.009 Small intestine mucosa 0.628 6.092 5.308 0.628 0.461 0.464 0.2260.143 Spleen 0.001 0.056 0.081 0.044 0.054 0.04 0.03 0.013 Stomachmucosa 0.027 0.187 0.235 0.079 0.12 0.086 0.067 0.041 Testis <0.002 0.020.052 0.037 0.061 0.057 0.063 0.045 Thymus <0.001 0.007 0.012 0.0080.014 0.012 0.009 0.005 Thyroid gland <0.001 0.001 0.002 0.001 0.0010.001 0.001 <0.001 n.d. = no data

TABLE 21 Time after administration (hrs) EPA-LPC 0.5 3 8 24 72 96 168 (%of dose/organ) Adrenal gland 0.002 0.014 0.023 0.042 0.03 0.018 0.012Blood 0.44 1.89 1.16 0.542 0.342 0.374 0.238 Bone <0.016 0.25 0.279 0.070.129 0.165 0.118 Bone marrow 0.007 0.118 0.189 0.197 0.124 0.137 0.055Brain 0.001 0.031 0.023 0.052 0.152 0.27 0.251 Eye (whole) <0.001 0.0020.002 0.001 0.002 0.003 0.003 Fat (white) 0.017 2.92 2.36 2.68 10.7 5.013.94 Kidney (whole) 0.034 0.237 0.307 0.344 0.304 0.242 0.19 Largeintestine mucosa 0.01 0.201 0.463 0.259 0.266 0.212 0.137 Liver 0.86413.4 12.4 8.24 5.23 4.35 2.56 Lung 0.043 0.191 0.291 0.198 0.182 0.1670.129 Muscle 0.106 4.01 7.14 8.16 8.48 10.5 8.41 Myocardium 0.023 0.2350.107 0.085 0.133 0.188 0.145 Pancreas 0.024 0.279 0.339 0.369 0.3020.293 0.257 Pituitary gland <0.001 0.001 <0.001 0.001 0.001 0.001 0.002Prostate gland <0.001 0.005 0.006 0.007 0.007 0.011 0.005 Skin 0.0211.26 1.01 0.986 1.55 1.51 1.36 Small intestine mucosa 28.4 10.2 3.132.23 0.861 0.542 0.389 Spleen 0.01 0.259 0.247 0.121 0.086 0.094 0.049Stomach mucosa 0.259 0.287 0.23 0.211 0.161 0.158 0.107 Testis <0.0020.04 0.054 0.077 0.069 0.093 0.081 Thymus 0.001 0.018 0.019 0.016 0.0190.023 0.016 Thyroid gland <0.001 0.003 0.002 0.002 0.003 0.003 0.002

TABLE 22 Time after administration (hrs) 0.5 3 8 24 72 96 168 336 DHA-PCAdrenal gland <0.001 0.007 0.008 0.005 0.009 0.01 0.004 0.005 % ofdose/tissue Blood 0.084 0.692 0.431 0.18 0.34 0.341 0.143 0.156 Bone<0.017 0.202 0.305 0.068 0.166 0.331 0.134 0.0235 Bone marrow <0.0010.042 0.047 0.035 0.061 0.078 0.028 0.021 Brain <0.001 0.015 0.02 0.0270.134 0.176 0.109 0.228 Eye (whole) <0.001 0.002 0.002 0.002 0.006 0.010.004 0.008 Fat (white) 0.052 3.976 2.57 1.892 5.701 5.942 2.365 2.691Kidney (whole) 0.011 0.156 0.154 0.091 0.205 0.287 0.101 0.091 Largeintestine mucosa <0.002 0.191 0.134 0.125 0.2 0.165 0.084 0.08 Liver0.222 7.552 5.905 2.814 3.9 3.875 1.291 1.124 Lung 0.007 0.113 0.0760.046 0.096 0.108 0.047 0.04 Muscle 0.111 6.575 6.166 2.432 8.408 14.8795.504 10.033 Myocardium 0.005 0.363 0.197 0.133 0.307 0.41 0.149 0.172Pancreas <0.001 0.199 0.15 0.066 0.162 0.188 0.067 0.085 Pituitary glandn.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. Prostate gland n.d. n.d. n.d.n.d. n.d. n.d. n.d. n.d. Skin 0.022 0.477 0.392 0.219 1.212 2.518 0.951.399 Small intestine mucosa 1.801 5.223 2.541 0.492 0.545 0.612 0.230.19 Spleen 0.002 0.058 0.045 0.027 0.046 0.053 0.017 0.016 Stomachmucosa 0.028 0.149 0.117 0.052 0.087 0.101 0.048 0.05 Testis <0.0020.016 0.018 0.025 0.082 0.083 0.047 0.069 Thymus <0.001 0.006 0.0060.004 0.013 0.013 0.006 0.005 Thyroid gland <0.001 0.001 0.001 <0.0010.001 0.001 0.001 <0.001 n.d. = no data

TABLE 23 Time after administration (hrs) 0.5 3 8 24 72 96 168 DHA-LPCAdrenal gland 0.003 0.014 0.026 0.011 0.015 0.035 0.012 % of dose/tissueBlood 0.421 2.1 0.674 0.315 0.358 0.364 0.202 Bone <0.017 0.232 0.1230.189 0.304 0.219 0.064 Bone marrow 0.005 0.129 0.167 0.112 0.14 0.130.106 Brain 0.001 0.027 0.058 0.077 0.247 0.333 0.441 Eye (whole) <0.0010.001 0.001 0.001 0.002 0.007 0.004 Fat (white) <0.017 1.49 3.8 12.26.59 5.57 3.79 Kidney (whole) 0.022 0.27 0.433 0.337 0.396 0.422 0.181Large intestine mucosa 0.008 0.202 0.399 0.146 0.3 0.29 0.19 Liver 1.5119.2 15.1 11.3 7.49 6.9 2.99 Lung 0.034 0.23 0.157 0.166 0.143 0.1640.114 Muscle 0.107 4.96 12.8 8.47 14.1 21.5 16.6 Myocardium 0.034 0.7560.442 0.404 0.418 0.643 0.532 Pancreas 0.014 0.413 0.33 0.332 0.3310.416 0.208 Pituitary gland <0.001 0.001 0.001 0.001 0.001 0.002 0.003Prostate gland <0.001 0.002 0.013 0.006 0.008 0.012 0.012 Skin 0.0420.512 1.09 1.17 1.52 1.75 2.1 Small intestine mucosa 26 4.32 5.25 2.291.18 0.951 0.416 Spleen 0.006 0.151 0.13 0.091 0.109 0.104 0.043 Stomachmucosa 0.046 0.268 0.319 0.186 0.145 0.139 0.072 Testis <0.002 0.0270.062 0.032 0.095 0.115 0.081 Thymus 0.001 0.013 0.016 0.011 0.018 0.020.013 Thyroid gland <0.001 0.002 0.002 0.002 0.002 0.003 0.001

1. A method for making a lysophosphatidylcholine (LPC) composition witha high content of EPA and DHA from a marine raw material containingphospholipids comprising treating the marine raw material with aphospholipase that is not native to the marine raw material to provide aphospholipase treated raw material and fractionating the phospholipasetreated raw material to provide an LPC composition having a higherlysophosphatidylcholine content than the starting raw material.
 2. Themethod of claim 1 wherein the raw material is selected from the groupconsisting of a krill lipid preparation, a herring lipid preparation, aherring roe lipid preparation, an algal lipid preparation, and a Calanuslipid preparation.
 3. The method of claim 1, wherein the krill lipidpreparation is a Euphausia superba lipid preparation.
 4. The method ofclaim 1, wherein the raw material is contacted with a phospholipase in asolvent.
 5. The method of claim 1, wherein the solvent is a mixture ofwater and an alcohol.
 6. The method of claim 5, wherein the alcohol isethanol.
 7. The method of claim 5, wherein the raw material is contactedwith a phospholipase in a mixture of about 85% water and 15% ethanol. 8.The method of claim 1, where the enzyme is a phospholipase A1 (PLA1). 9.The method of claim 1, wherein the enzyme is a phospholipase A1 (PLA1)and wherein the enzyme concentration is in the range of 0.1-20 vol/wt %.10. The method of claim 1, wherein the enzyme is a phospholipase A1(PLA1) and wherein the method is carried out at a pH of 3-12.
 11. Themethod of claim 1, wherein the enzyme is a phospholipase A1 (PLA1),wherein the method is carried out between 4-95° C.
 12. The method ofclaim 1, wherein the raw material has a content of EPA and DHA in therange of EPA: 1-70 wt % and DHA: 1-70 wt %.
 13. The method according toclaim 1, wherein the LPC composition has a content of EPA and DHA in therange of EPA: 5-100 wt % and/or DHA: 5-100 wt %.
 14. The methodaccording to claim 1, wherein the LPC composition has an LPC content inthe range of 10-100 wt %.
 15. The method of claim 1, wherein the LPCcomposition obtained by the process is characterized in comprising fromabout 20% to about 100% LPC w/w of the composition and an omega-3 fattyacid content of from 5% to 50% w/w of the composition, a ratio ofEPA:DHA of from 1:1 to 3:1 on a w/w basis or a ratio of DHA:EPA of from1:1 to 5:1 on a w/w basis, and a 2-LPC:1-LPC ratio of from 1:8 to 18:1on a w/w basis.
 16. The method of claim 1, further comprising the stepof concentrating the enzyme-treated lipid composition to provide aconcentrated LPC composition.
 17. The method of claim 16, wherein theLPC composition obtained by the process is characterized in comprisingfrom about 40% to about 100% LPC w/w of the composition and an omega-3fatty acid content of from 5% to 50% w/w of the composition, a ratio ofEPA:DHA of from 1:1 to 3:1 on a w/w basis or a ratio of DHA:EPA of from1:1 to 5:1 on a w/w basis, and a 2-LPC:1-LPC ratio of from 1:8 to 18:1on a w/w basis.
 18. The method of claim 16, wherein the concentratingcomprises phase separation of the LPC composition with solvents ofdifferent polarity to provide a concentrated LPC composition.
 19. Themethod of claim 16, wherein the concentrating comprises chromatographicseparation of the LPC composition to provide a concentrated LPCcomposition.
 20. The method of claim 1, further comprising formulatingthe LPC composition for human consumption.